Evaluation of different approaches to protein engineering and modulation

EVALUATION OF DIFFERENT APPROACHES TO PROTEIN ENGINEERING AND MODULATION APARNA GIRISH (M.Sc. (Hons), BITS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGMENTS My two and half year research in science has been an eye opening experience. Before I went into research, science had always been awe inspiring from far, from the text books. My masters has taught me that behind the awe inspiring discoveries lies a lot a hard work from large teams of dedicated and zealous scientists. Putting theory into practice has certainly been challenging. Trouble shooting becomes a way of life in the lab, it brings forth opportunities to learn more. I’m glad that the journey through science has been a rewarding and a great learning experience for me and all that would not have been possible but for a bunch of people whom I owe this acknowledgement to. I would like to thank my supervisor, Prof. Yao Shao Qin, for his ideas, for constantly trying to bring forth the best in me, for never giving up, for the motivation and for the guidance throughout my projects. I thank all my lab mates for their constant support and valuable suggestions. I would also like to thank the graduate committee of the Department of Biological sciences, for having given me this opportunity to learn and do science in NUS. Lastly but certainly not the least, I thank mother nature, for being so diverse, intricate, complex and beautiful, so that humans in their life time on earth may never be short of discovering and experiencing the true joy that only science can bring. i TABLE OF CONTENTS Acknowledgements i Table of contents ii Summary vii List of publications ix List of tables x List of figures xi List of abbreviations xiii 1. Introduction 1.1 1.2 1 Protein engineering 1 1.1.1 Rational design and protein evolution to create novel functions or improve existing functions. 1 1.1.2 Protein engineering: introducing artificial functionalities using enzyme mediated approaches 2 1.1.3 The three different approaches to protein engineering and modulation that were evaluated in this report 3 Inteins 1.2.1 1.3 4 Mechanism of protein splicing 5 1.2.2 Engineered inteins in biotechnological applications 5 1.2.3 The intein based method to tag proteins site-specifically 7 Phage display 8 1.3.1 Applications of phage display 11 1.3.2 Enzyme evolution on phage 14 1.3.2.1 Developing a strategy to evolve SrtA on T7 phage 14 ii 1.3.3 2. Affinity selection of binders against 3CL protease mutant from SARS 19 Materials and methods 22 2.1 Making chemically competent bacteria for transformation 22 2.2 Transformation of plasmids/ligated vectors into chemically competent cells 22 2.3 Transformation of yeast cells 23 2.4 PCR 23 2.5 Cloning 24 2.5.1 TA cloning 24 2.5.2 Gateway cloning 24 2.5.3 RE-based cloning into conventional plasmids and large bacteriophage genomes 26 2.6 Sequencing of genes 28 2.7 Site directed mutagenesis of genes 28 2.8 Expression of different fusion proteins from different vectors and hosts. 30 2.9 Western blot of proteins 32 2.10 Affinity chromatography of proteins 33 2.10.1 Ni-NTA column 33 2.10.2 GSH column 34 2.10.3 Chitin column 35 2.11 Production of N-terminal cysteine proteins 35 2.12 Spotting of N-terminal cysteine proteins on thioester slides 36 2.13 In vitro biotinylation of proteins 37 2.14 Spotting biotinylated proteins onto avidin slides 37 iii 2.15 2.16 Enzyme activity assays 38 2.15.1 Testing activity of SrtA 38 2.15.2 In vitro self-ligation assay 39 2.15.3 Self-ligation assay on the phage 39 General phage methods 40 2.16.1 Packaging of T7 phage DNA 40 2.16.2 Amplification of phages 40 2.16.3 PEG precipitation of phages 41 2.16.4 Storage, Serial dilution and titering of phages 41 2.16.5 Plaque lift 42 2.16.6 Sequencing phage 43 2.16.6.1 M13 43 2.16.6.2 T7 43 2.17.7 Phage enrichment methods 3. 44 2.17.7.1 Affinity based enrichment of C-SrtA-T7 44 2.17.7.2 Activity based enrichment of C-SrtA-T7 44 2.17.7.3 Bio-panning against SA and 3CL mutant 45 2.17.7.4 Binding assay 46 Results and discussion 47 3A The intein mediated approach to site-specifically label proteins 48 3A.1 The intein based method to produce N-terminal cysteine proteins 48 3A.1.1 Expression of N-terminal cysteine -containing proteins from bacteria. 49 iv 3A.2 3B 3C 3A.1.2 Spotting N-terminal cysteinecontaining EGFP onto thioester slides 49 The intein mediated method to site -specifically label proteins derived from yeast 50 3A.2.1 Expression levels and the in vivo cleavage pattern of the Intein-fusion proteins in yeast 52 3A.2.2 On-column cleavage and generation of biotinylated proteins 54 3A.2.3 Detection on the microarray 56 Designing a selection scheme to evolve SrtA on phage 58 3B.1 The N-terminus extension scheme 58 3B.2 The C-terminus extension scheme 60 3B.3 Activity of SrtA with N-and C-terminal extensions 60 3B.4 Self-ligation assay of N/C-SrtA 62 3B.5 Display of N/C-SrtA on phages 62 3B.6 Activity assay of the SrtA on phage 64 3B.7 Enrichment of SrtA-phages from a pool of bare phages 67 3B.7.1 Affinity based enrichment 67 3B.7.2 Activity based enrichment 67 Detection of binders of 3CL protease from a phage library 71 3C.1 Biopanning of a model protein Streptavidin 71 3C.2 Binding assay to detect the strongest of binders 72 3C.3 Expression and mutation of the 3CL protease 74 3C.4 Bio-panning against the 3CL protease mutant 74 4. Conclusions 79 5. References 82 Appendix A 95 v Appendix B 102 vi SUMMARY Proteins are important molecular machines within cells. Ability to modulate and engineer proteins serves as important tools to understand their structure and function. Different methods are available to engineer proteins. These include protein evolution methods and enzyme-based methods to introduce artificial functionalities. Protein evolution can give rise to useful proteins that can fulfill biotechnological and industrial applications. Protein engineering methods which add on small molecule tags site-specifically have many applications including bio-imaging, where by specifically adding a fluorescent tag onto a protein, one can study protein dynamics, localization, cell movement and cell growth. Site-specific modification of proteins has also found use in the field of microarrays, where adding on tags such as biotin to a protein allow it to be specifically immobilized onto an avidin-coated surface. Different approaches to protein engineering and modulation using the phage display method and the intein splicing strategy were evaluated in this report. A strategy for the immobilization of proteins site-specifically via the N-terminus onto the microarray was developed. The chosen model proteins were cloned into a vector system that facilitates the expression of the protein with an N-terminal intein fusion. An extra cysteine residue was introduced at the junction of the intein and protein fusion. Upon expression of the intein-protein fusion, intein splices out leaving the protein with an N-terminal cysteine. The proteins thus produced can then be applied to thioester-functionalized slides for uniform orientation. As a complementary approach, a system to biotinylate the C-terminus of proteins derived from yeast was set up. The expression levels and the splicing patterns of three different intein fusion vii constructs were studied. Optimal conditions for biotinylation of a model protein were achieved and the immobilization efficiency onto to an avidin microarray was evaluated. As an approach to protein engineering for the enzyme Sortase, a selection scheme for the evolution of increased activity of Sortase on phage has been devised. Sortase is a transpeptidase, which catalyzes the transfer of N-terminal glycine peptides to the sorting motif LPETG found in proteins. Studies of Sortase revealed that it could be used for attaching small molecule tags to proteins and that Sortase is not a very robust enzyme in vitro. A selection scheme has been devised to select for mutants of Sortase with improved activity by displaying them on the surface of the phage. Using this selection method and a suitable screening system, Sortase may be evolved into a more active enzyme. Phage display library displaying random peptides was scanned for good binders to the active site mutant of SARS main protease 3CL. Using the affinity selection method in phage display, multiple rounds of selection were carried out. A binding assay at the end selection revealed the existence of weak binders to the protease. Several candidate peptides that bound the mutant protease with low affinity were sequenced and identified. viii LIST OF PUBLICATIONS 1. Girish, A., Chen, G.Y.J., and Yao,S.Q., (2006) “Protein engineering for surface attachment”in Microarrays:pathways to drug discoverey. (P.predki, ed.) CRC press. 2. Girish, A., Sun, H., Yeo, D.S.Y., Chen, G.Y.J., Chua, T.-K. and Yao, S.Q. (2005), Site-specific immobilization of proteins in a microarray using inteinmediated protein splicing. Bioorg. Med. Chem. Lett.,15, 2447-2451. 3. Zhu, Q., Girish, A., Chattopadhaya, S., and Yao, S.Q., (2004), Developing novel activity-based fluorescent probes that target different classes of proteases. Chem. Commun., 1512-1513. ix LIST OF TABLES 1. Results from the binding assay from biopanning against SA 73 2. Sequencing results of the peptides from biopanning against SA 75 3. Sequencing results of the peptides from biopanning against Streptavidin 76 4. Sequencing results of the peptides from biopanning against 3CL mutant 78 x LIST OF FIGURES 1. Mechanism of protein splicing 6 2. The intein-mediated strategy to biotinylate proteins at the C-terminus 10 3. General scheme for affinity based enrichment of peptides libraries on phage 13 4. Hydrolysis and transpeptidation activity of Sortase 16 5. Evolution of SrtA on phage 8 6. Self ligation of G5-BIOTIN substrates onto C-SrtA 16 7. Self ligation of biotin-LPETG substrate onto N-SrtA 20 8. Self ligation of G2-TMR substrates onto C-SrtA displayed on the T7 phage 20 9. The BP cloning reaction 25 10. The LR cloning reaction 25 11. RE-based cloning of SrtA into the T7 phage genome 29 12. Overview of site-directed mutagenesis methods 31 13. Results from N-terminal immobilization strategy 51 14. Native fluorescence of EGFP-Intein fusion from yeast after cell lysis and clarification 53 15. Expression timeline of the three EGFP-Intein-CBD fusions in the yeast host detected using anti-CBD western blot 53 16. In vivo cleavage pattern of the three EGFP-Intein fusions in yeast crude cell lysates as detected using anti-CBD western blot 55 17. Purification of EGFP-Intein 3 fusion from yeast small scale cultures 55 18. Effect of different concentrations of cys-Biotin on the biotinylation efficiency of EGFP purified from different hosts 55 19. Purification of SrtA and different versions of SrtA 63 20. Activity assay of SrtA, N-SrtA and C-SrtA as detected by in-gel fluorescence scanning 63 xi 21. Transpeptidation of SrtA and C-SrtA as measured using quenched fluorescent substrates 65 22. Hydrolytic activity of SrtA and C-SrtA as measured using quenched fluorescent substrates 65 23. Self-ligation assay of N-SrtA and C-SrtA as detected by anti-Biotin western blot 66 24. Plaque lift 66 25. Expression levels and activity assay of C-SrtA on the T7 phage as detected by anti-Biotin western blot 66 26. Activity assay of C-SrtA on T7 phage 69 27. Self ligation assay of C-SrtA on T7 phage 69 28. Affinity enrichment of C-SrtA-T7-phage 69 29. Activity based enrichment of C-SrtA-T7-phage 70 30. Expression levels of 3CL protease and 3CL protease mutant 75 xii LIST OF ABBREVIATIONS A Alanine Amp Ampicillin BPB Bromo Phenol Blue C Cysteine CBD Chitin Binding Domain Cys-Biotin Cysteine – Biotin DABCYL α-(t-BOC)- -(4-DimethylAminophenylazoBenzoyl)-Llysine ( -(t-BOC)- -dabcyl-L-lysine) DBS Department of biological sciences Dil Dilution dNTP deoxy Nucleotide Tri Phosphate DNA deoxy Nucleic Acid DTT Di Thio Thrietol E Glutamic acid EDTA Ethylene Diamine Tetra Acetic acid EGFP Enhanced Green Fluorescent Protein ELISA Enzyme Linked Immuno Sorbent Assay F Phenyl alanine FITC Fluorescein Iso Thio Cyanate Fwd Forward Gly (G) Glycine GSH Glutathione GST Glutathione S Transferase xiii His (H) Histidine I Isoleucine IPTG IsoPropyl-beta-D-Thio-Galacto-pyranoside kDa kilo Daltons L Leucine LB Luria Bertani Min Minute(s) MESNA Methyl Ethyl Sulfonic Acid Ni-NTA Nickel- Nitrilo Tri Acetic acid NUS National university of Singapore NEB New England Biolabs O/N Over Night (12 hours) OD Optical Density ORF Open Reading Frame P Proline PBST Phosphate Buffered Saline with Tween-20 PCR Polymerase Chain Reaction pfu Plaque Forming Units PEG Poly Ethylene Glycol PVDF Poly Vinidiliene Di Fluoride PAGE Polyacryl Amide Gel Electrophoresis Q Asparagine RT Room Temperature RE Restriction Enzyme Rev Reverse xiv RNA Ribo Nucleic Acid S Serine SARS Severe Acute Respiratory Syndrome SrtA Sortase A Sec Seconds SDS Sodium Dodecyl Sulphate SA Streptavidin SH3 Src like Homology TMR tetra methyl rhodamine T Threonine U Units V Valine W Tryptophan X-Gal 5-bromo-4-chloro-3-indolyl- beta -D-galactopyranoside Y Tyrosine 2XYT Rich growth media, see appendix for composition 6XHIS Poly Histidine (6 repeats of Histidine) 2-ME 2-Mercapto-Ethanol 3CL 3C like xv 1. INTRODUCTION 1.1 Protein engineering Proteins are the most important work horses in the cells; they serve myriad functions and are also important structural determinants within cells. Ability to modulate and engineer proteins serves as important tools to understand their structure and function [1], it can also give rise to useful proteins that can fulfill biotechnological and industrial applications [2]. The terms “Protein engineering” and “modulation” are used in the following context throughout the thesis and are defined as, “Processes of modifying the structure of proteins or introducing unnatural functionalities to create tailor-made proteins serving useful applications”. Several methods that exist to modify and engineer proteins can be broadly grouped into 2 different categories. (a) Rational design and Protein evolution methods to create novel functions or improve existing functions. (b) Enzyme-based methods to introduce unnatural but useful functionalities. 1.1.1 Rational design and protein evolution to create novel functions or improve existing functions. Proteins as such are pretty robust inside cells, but their performance is typically hampered outside natural environments and several proteins fail to behave well in industrial applications [2, 3]. Traditionally the approach to study and design proteins with improved or novel function has been through the genetic method of site directed mutagenesis [1]. It requires detailed knowledge of protein structure and has the limitation in that substitution of desired amino acids can be done only with their natural amino counterparts. Proteins are complex entities and more often it is very difficult to predict exactly what structural changes will give rise to the desired 1 function. These limitations can be overcome by taking the proteins through the process of protein evolution [4], which mimics the natural process of evolution in the laboratory test tube. The key points of the protein evolution methods are mutagenesis and selection of the fittest. A repertoire of random mutants of a desired gene is created using genetic methods like error prone PCR or gene shuffling and linked to a suitable genetic coding system like phage display. The pool of mutants is then passed through a selection/screening assay that select for the mutants with the desired function. The genetic pool is culled periodically of undesirable mutations through a negative selection if possible. The whole process of mutagenesis and selection/screen may then be repeated until the proteins with desirable functions evolve [5]. Thus it is in essence bringing natural evolution to the test tube. 1.1.2 Protein engineering : introducing artificial functionalities using enzymemediated approaches Several enzymes that can site specifically add on small molecule functionalities have been exploited to modify proteins. Some of them include Inteins [6-11], Biotin ligases [12], Sortase [13], Sfp phosphopantetheinyl transferase [14] and Amino Acyl - tRNAtransferases [15-19]. Protein engineering methods which add on small molecule tags site specifically have many applications. One such example is in the field of bioimaging, where by specifically adding on fluorescent tags onto proteins, one can study protein dynamics, localization, cell movement and cell growth [20, 21]. Site-specific modification of proteins has also found use in the field of microarrays, where adding on tags like biotin to a protein allows it to be specifically immobilized onto an avidincoated surface [10, 11, 22]. 2 1.1.3 The three different approaches to protein engineering and modulation that were evaluated in this report In this report, three different approaches to protein engineering and modulation were evaluated. As one of the approaches to protein engineering, a strategy for the immobilization of proteins site-specifically via the N-terminus onto the microarray was developed. The chosen model proteins were cloned into a vector system that facilitates the expression of the protein with an N-terminal intein fusion. An extra cysteine residue was introduced at the junction of the intein and protein fusion. Upon expression of the intein-protein fusion, intein splices out, leaving the protein with an N-terminal cysteine. The proteins thus produced can then be applied onto thioesterfunctionalized slides for uniform orientation. As a complementary approach, a system to biotinylate the C-termini of proteins derived from yeast was set up. The expression levels and the splicing patterns of three different intein fusion constructs were studied. Optimal condition for biotinylation of a model protein was achieved and the immobilization efficiency onto to an avidin microarray was evaluated. Once the system was established it was foreseen that important enzymes present in the yeast namely the kinases, phosphatases and proteases could be immobilized using this versatile method to generate an enzyme array. The enzymes could then be studied in a high throughput fashion using some of the available activity-based fluorescent probes in our lab [23-26]. As an approach to protein engineering, a selection scheme for the evolution of increased activity of SrtA on phage has been devised. SrtA is a transpeptidase, which catalyzes the transfer of N-terminal glycine peptides to the sorting motif LPETG found in proteins. Studies of SrtA revealed that it could be used for attaching small 3 molecule tags to proteins and that SrtA is not very robust in vitro. A selection scheme has been devised to select for mutants of SrtA with improved activity by displaying them on the surface of the phage. Using this selection method and a suitable screening system, SrtA could be evolved into a more active enzyme. Phage display library displaying random peptides was scanned for good binders to the active site mutant of SARS main protease 3CL. Using the affinity selection method in phage display, multiple rounds of selection were carried out. A binding assay at the end of multiple rounds of selection revealed the existence of weak binders to the protease. Several candidate peptides that bound the mutant protease with low affinity were sequenced and identified. The subsequent sections of this chapter will introduce some of the relevant topics in more detail. 1.2 Inteins Inteins are naturally occurring in frame protein fusions that can self splice out, ligating together the extein sequences of the gene in which they occur. They are very similar to the group I self splicing introns which splice at the RNA level [27]. Inteins since their first description in yeast Saccharomyces cerevisiae [28, 29], have now been identified in all three kingdoms of life, as well as in bacteriophages. Many of the inteins like the group I introns are mobile at the genetic level because they code for homing endonucleases [27]. Upto 70% of the inteins identified are found in genes that are related to DNA metabolism including DNA polymerases, helicases and gyrases [27], which often are vital genes to the organism [30]. Although inteins have been denoted as selfish genes, because no known function exists for many of them, some experiments have suggested that they might hold regulatory roles in cells, and that 4 ancestral inteins might have had some function but they were lost during evolution [27, 30]. 1.2.1 Mechanism of protein splicing The mechanism of protein splicing has been well studied by many groups [31-33]. There are several key residues involved in protein splicing. The first amino acid of Nintein (see Figure 1) is invariably a cysteine or a serine residue. The thiol or the hydroxyl side chains of these amino acids undergo an acyl shift at the N-terminal splice junction. The first residue of C-extein is invariably a cysteine or threonine or serine. The side chains of these are equipped to attack the electrophilic N-terminal splice junction, resulting in a branched intermediate. An asparagine residue precedes the C-terminal splice junction, and helps in resolving the intermediate through cyclization and succinimide formation. Eventually an S-N acyl shift releases the spliced product. 1.2.2 Engineered inteins in biotechnological applications Intein splicing does not require cofactors and the process of splicing is very efficient [34]. The novelty of inteins, ever since their discovery, has been exploited for a variety of biotechnological applications, ranging from synthesizing toxic proteins (by expressing them in two parts in the cell and ligating them externally using the inteinmediated method to give the native peptide bond [35]), cyclization of proteins and peptides [36], attaching novel functionalities to proteins site specifically [6-11], generation of novel protein combinations [37] and intein mediated genetic switches 5 Figure 1: The steps involved in the self splicing of inteins, see text for details. (Splicing mechanism taken from http://www.neb.com/neb/inteins.html) 6 [38, 39]. NEB has commercialized vectors that enable the cloning of desired genes with an intein either at the N/C-terminus and a CBD tag. Upon expression and affinity column purification, the protein of interest can be cleaved off by inducing intein cleavage under some specified conditions [31]. The inteins that can splice out conditionally were engineered from the native counterparts through a combination of both rational engineering and directed evolution approaches [40-42]. These inteins were designed such that they splice out only from either N/C-terminus, and they were pH or thiol agent inducible [40-42]. 1.2.3 The intein based method to tag proteins site-specifically Protein microarray is emerging as a powerful tool in the high throughput analysis of protein abundance and function [43-45]. One of the key concerns in the fabrication of functional protein microarrays is the method of immobilization, which to some extent determines whether or not a protein retains its function [46]. There are two obvious choices, either random immobilization or methods that allow site-specific uniform orientation. Both of these methods have been used to develop protein microarrays [47]. In this report a strategy for the immobilization of proteins onto a microarray site specifically via the N-terminus was developed. For the N-terminal immobilization, the chosen model proteins were cloned onto a vector system that facilitates the expression of the protein with an N-terminal intein fusion. An extra cysteine residue was introduced at the junction of the intein and protein fusion. Upon expression of the intein-protein fusion, intein splices out, leaving the protein with an N-terminal cysteine [6]. N-terminal cysteine containing EGFP was produced in this manner and successfully immobilized onto thioester glass surface. Also as a complementary approach to the N-terminal immobilization, immobilizing proteins expressed from a 7 yeast host, via a C-terminus biotin moiety onto avidin-functionalized microarrays was considered. Three different inteins available from NEB, were fused individually to the C-terminus of the EGFP, and expressed in a suitable yeast expression system. The expression levels and the in vivo cleavage pattern of the three inteins were analyzed. One of the three inteins, the Sce VMA Intein was found to express better than the other fusions and the in vivo cleavage of the fusion was minimal. Hence the Sce VMA Intein fusion was chosen for further studies. Fusion to intein at the C-terminus allows the production of thioester functionality at the C-terminus, which in turn can react with the sulfhydryl moiety of the cysteine in cysteine-biotin, to give a C-terminal biotinylated protein via a native peptide bond. Using this, a model protein EGFP was biotinylated and the immobilization efficiency onto to an avidin microarray was evaluated. Once the system was established it was foreseen that important enzymes present in the yeast, namely the kinases, phosphatases and proteases, could be immobilized in a high-throughput fashion using this method. Then they could be studied in a parallel fashion using the available activity based fluorescent probes in our lab [23-26]. 1.3 Phage display Bacteriophages are virus that feed on bacteria. They have a simple structure with their nucleic acid genome surrounded by a coat of proteins [50]. There are two kinds of bacteriophages, the lytic ones and the nonlytic ones [51]. For many years bacteriophage genomes have traditionally been used as vehicles of gene transfer to bacteria [51, 52]. The concept of phage display was introduced by George P Smith, who came up with a method to display foreign peptides on the surface of the bacteriophage M13, through a fusion to its coat protein Gene III [53]. Product of Gene 8 III resides on the tip of the filamentous bacteriophage and is involved in infection of the host bacterium. He found that small peptides fused to the N-terminus of the Gene III can be displayed on the phage tip without interference to its infective capacity [53]. He called his method phage display and demonstrated its first application in mapping epitopes of antigens [54]. Ever since, peptides and proteins have been successfully displayed on phage and a collection of phage display vectors are now commercially available [52]. Typically the protein/peptide of interest is cloned into either bacteriophage genome/phagemids using traditional cloning methods. The most commonly used phage is the M13 phage. There are two different genes that are typically used for the display of peptides and proteins on M13. One is the gene III, this is present in up to 5 copies on the tip of the virion. Gene VIII is another available display protein system, it is present in up to 2700 copies per virion. Due to steric limits only small peptides are tolerated in the latter system. The Gene III system can tolerate proteins up to 100kDa [3], but the number of copies displayed on each virion should be limited as the protein gets larger [55]. This is done by cloning the protein of interest into a phagemid rather than into the phage genome itself, and then rescuing the phagemid (a phagemid is a plasmid with phage origin of replication; when F+ cells, harboring such phagemids are infected by a helper phage, the phagemid gets replicated from the phage origin and packaged into the phage heads preferentially over the helper phage genome) using helper phages (phages whose genomes contain impaired origin of replications). When using the phagemid method to display proteins, depending on the size of the protein and how well it is tolerated on the phage, the number of copies of the displayed protein can vary from 0-5 per phage. 9 Intein fusion construct EGFP INTEIN CBD Transformation into yeast cells MESNA mediated cleavage and tagging of proteins with CysBiotin v A idin Capture onto chitin column Biotin + Slides functionalized with avidin vvv vvv A idin idin a idin idin A A idin a a idin Immobilization of C-terminal biotinylated proteins onto microarray vvv vvv A idin idin a idin idin A A idin a a idin Figure 2: The intein mediated strategy to Biotinylate proteins on the C-terminus. Intein is fused to the C-terminus of the desired protein using recombinant DNA methods. Upon protein expression, the fusion protein is pulled down onto chitin beads. When incubated with MESNA (a thiol reagent that induces splicing of intein), the sulfydryl group of Cys-Biotin attacks the thioester intermediate that is formed at the C-Terminus of the protein to give a covalent native peptide bond. The desired protein is thus biotinylated. 10 1.3.1 Applications of phage display Using the method described above, libraries of peptides or proteins can be displayed on the phage giving rise to a number of applications [55]. The phage can be then viewed as a huge bead with a protein/peptide of interest tethered to it. The genetic information of the protein/peptide resides inside the phage and is retrievable any time by a simple sequencing step. As such the phage then is a coded, amplifiable and infinitely storable bead. In the affinity selection method, the protein of interest is coated onto a solid surface and the phages bearing the random peptide libraries are applied to it. After incubation, the non binders are washed off, and the binders are eluted by nonspecific methods that disrupt protein-protein interaction (e.g. glycine at pH2.2, the phages themselves are extremely robust and can withstand harsh chemical conditions) or by the use of a known competing ligand (see Figure 3). Then the binders are amplified and enriched, before going through another round of selection. The selection rounds are repeated until significant binders emerge. The binders are identified through DNA sequencing. Typically a consensus sequence emerges (a group of binders with similar sequences). To cite a few interesting examples, using the method of affinity selection, a number of cloned SH3 domains were used to select ligands from a random peptide library. Upon identification of the ligands, these were used to probe conventional cDNA libraries for protein that bind to the identified ligands. In this manner 18 homologs of the SH3 domain were identified, several of them previously unknown [63-65]. A peptide mimic of the natural protein hormone erythropoietin [66] has been identified using this method. L-amino acid peptide ligands for the D-amino acid isoform of the SH3 11 domain have been selected. The D-version of the L-peptides, then are ligands for the natural SH3 protein [67]. Proteolysis is a common form of posttranslational modification and is important in several biological cascades and signaling pathways [68]. Knowledge of protease specificity allows us to design better inhibitors, identify biologically relevant substrates and is useful in applying proteases in site-specific proteolysis. Substrate characterization of a protease is a time consuming step with traditional methods, which involve scanning of peptide libraries or deriving substrates from physiological substrates. After the introduction of phage display by Smith and colleagues, a method called “substrate phage” came into use for the discovery of substrates of proteases [69-75]. In this method, random peptide libraries which represent potential substrates of a protease are displayed on the surface of the phage. One end of the substrate is tethered to the phage while the other end is fused to any convenient affinity domain. Following immobilization of the substrate phages on the affinity support, the phage is incubated with the protease whose substrate specificity is to be determined. Only potential substrates will be released, which can then be amplified to increase their number and subjected to further rounds until good substrates emerge. 12 Library of phages bearing peptides on the surface Incubation of the of phage with the receptor Affinity based binding of ligand phages to the receptor Immobilized receptor The process is repeated until dominant binders emerge Bound phages are eluted using a known affinity ligand Unbound phages are washed away Eluted phages are amplified by infection with a host bacterium Figure 3: General scheme for affinity based enrichment of peptide libraries on phage. 13 1.3.2 Enzyme evolution on phage Enzymes have been displayed on the surface of phages in order to be evolved into more active, or more stable counterparts or into mutants recognizing different susbstrates [3-5, 76]. Evolution of enzymes on phage, apart from the requirement of active display on the phage also requires a good selection scheme which can select for the active members from the library of mutants displayed on the phage. To date, several such selection strategies have been employed to evolve enzymes on phage [77-86]. One very interesting selection scheme is the product capture approach. This was introduced simultaneously and independently by two different groups [81, 82]. The enzyme is displayed on the phage, and alongside the enzyme the substrate is displayed in close proximity (either chemically ligated to the surface coat proteins of the phage [82], or ligated by means of electrostatic interaction, followed by a chemical crosslink [81]). Thus the substrate is accessible to the enzyme active site, now active enzymes are able to convert substrate to product. The next step is product-capture and it involves capture of the reaction product by a product-specific reagent or antibody. In this report a selection scheme for the evolution of active mutants of SrtA on phage has been devised. 1.3.2.1 Developing a strategy to evolve SrtA on T7 phage SrtA is one of the homologs of the transpeptidase Sortase discovered in gram positive bacteria [87]. It catalyses a transpeptidation reaction that anchors proteins important for the pathogenesis of gram positive bacteria to their cell wall [88]. The crystal structure of SrtA has been solved [89]. The sorting mechanism has been well studied 14 [90-96]. Proteins bearing the signature motif LPETG (a 5 mer peptide) are cleaved by SrtA between T and G and ligated to N-terminal glycine containing peptidoglycan building unit. Thus proteins that are important for the pathogenesis are sorted and attached onto the cell wall covalently. It has been proposed that SrtA might be a good drug target against gram positive bacteria [97]. The protein has been purified, with its membrane anchor removed [98], (N terminal 60 amino acids) and its kinetics has been well studied [99]. According to a HPLC assay the kinetic parameters have been established as Km = 5.5 mM for the LPETG substrate and 140 µM for the glycine substrate [99]. It has been shown that Gn, n = 1 to 5 can be used as nucleophilic substrate mimic of SrtA. Sortase has also been viewed as an attractive target enzyme to carry out modifications of proteins [100] such as ligating specific tags to the terminus of a protein [13], and as a self cleavable affinity tag for affinity purification of proteins [101]. Here in this project it was hypothesized that SrtA could be used to ligate fluorescent probes to proteins engineered to have a LPETG motif, and ultimately be useful for imaging proteins in live cells. To this end it was shown that EGFP protein expressed with a LPETG motif at its C-terminus could be successfully ligated with GG-TMR. Fluorescent probes were designed to study the hydrolysis and transpeptidation activity (see Figure 4). Although SrtA has been proven to be a very robust enzyme inside the cell (can sort proteins within min inside the cells [88]) it is not very active in vitro with a modest Kcat of 0.27/sec [99]. Upon transferring SrtA into the mammalian cell the activity of SrtA which has a bacterial origin might not be optimal. Hence it was decided to evolve SrtA into a much more active enzyme. With this goal in mind and based on a strategy similar to the product capture approach of Subtiligase [102], 15 Quenched fluorescent substrate Hydrolysis SH A Q L A H2O T Transpeptidation T G L E P E A P GG NH2 A-ACC Q- DABCYL T L E GG + G + COOH Q P G Figure 4: Hydrolysis and transpeptidation activity of SrtA. To detect the hydrolysis activity of SrtA, it was incubated with a quenched fluorescent substrate (ACCLPETG-DABCYL), upon cleavage of the T-G bond, the fluorescence of ACC is released. SrtA solely catalyses a transpeptidation activity in the presence of a nucleophilic GG-substrate, thus when incubated with the quenched fluorescent substrate and GG-peptide, SrtA mediates the transfer of GG-peptide to the substrate thus releasing the florescence of ACC. SH LPET↓G-COOH + H 2N-G5 - Sortase A - Biotin SH LPET-G5 + HOOC-G Figure 6: Self-ligation of G5-BIOTIN substrates onto C-SrtA. The substrate LPETG was fused to the C-terminus of SrtA (C-SrtA). Upon incubation with the pentaglycine substrate conjugated to biotin, SrtA is able to self-ligate the biotin substrate onto itself. 16 we decided to display mutants of SrtA on the surface of phage, and select for active members using a self ligation scheme (see Figure 5). In the self ligation scheme the N- or C-terminus of SrtA is extended to include the corresponding substrates of SrtA (SrtA as mentioned previously needs two substrates, a LPXTG peptide and NH2-Gn =15 with the NH2 termini free for nucleophilic attack). Accordingly the LP(X=E)TG – COOH substrate was fused to the C-terminus of SrtA, which will be called C-SrtA, a NH2-(GGGSE)3 substrate was fused to the N-terminus of SrtA, which will be called N-SrtA (see Figures 6 and 7). Substrate fusion on SrtA was done and the activity and self-ligation ability of SrtA was tested. It was shown successfully that the concept of self-ligation worked on free SrtA on both the display systems. Following this the N-SrtA and C-SrtA were displayed on phage and the activity was tested. Display on the M13 phage allows the N-terminus of the displayed protein to be free. Display on the T7 phage allows the C-terminus of the displayed protein to be free. For display onto the M13 phage, SrtA was fused to the N-terminus of gene III. To display proteins on the T7 phage, SrtA was fused to the C-terminus of the capsid gene10B. While we could conclusively see that the SrtA on T7 phage was active after display, we failed to prove activity of SrtA on the M13 phage. Following this all experiments used the T7 phage system only. C-SrtA-T7 could be successfully affinity-purified from a pool of non-SrtA phages. Additionally, for the selection system to work, it was required to prove that the self-ligation assay works on the phage as well. Towards this end, we proved that SrtA on T7 phage could successfully carry out the self-ligation of GG-TMR onto itself (see Figure 8). Thus a selection 17 CAPSID SORTASE Active members ligate substrate onto themselves 6X-HIS LPXTG G G T G L G G E L G G G P A mutant library of Sortase is made Incubated with GG-Biotin bound SA beads E PL T G T G E P Displayed on phage The whole process is repeated until the desired activity is obtained Ni-2+ T G L E P Inactive members are pulled down via the HIS tag The active members are amplified Figure 5: Evolution of SrtA on phage. 18 method was successfully designed, with which one may be able to select in future, from a pool of random mutants of SrtA, the active members. 1.3.3 Affinity selection of binders against 3CL protease mutant from SARS The SARS coronavirus, the causative agent of Severe Acute Respiratory Syndrome [103], was sequenced and revealed to have 2 overlapping poly-proteins [104]. A 3C like protease encoded in the poly-protein was involved in cleaving the poly-protein to generate functional proteins responsible for the replication of the virus. Based on the sequences of the different strains of the SARS virus sequenced, the 3CL protease was highly conserved and it also shared homology with main proteases from other coronavirus [105]. The protein has been cloned and purified [106] and its 3D structure has been solved [107]. The cleavage preference of the protease lies in the P1, P2, and P1’ residues. It prefers a glutamine in the P1 position, hydrophobic residue in the P2 position and alanine, serine or glycine residue in the P1’ residue [106]. Given the fact that this is an important protease in the life cycle of the virus, the 3CL protease was considered to be an important drug target for SARS. A small molecule library, which also included some current drugs in the market (based on molecular simulation experiments these had previously been proposed to be inhibitors of the virus) was screened against the virus. Most of the predicted drugs had no effect on the virus, while some others from the library did show inhibition [100]. Given the fact that the 3CL protease was considered an important drug target and due to the paucity of the available inhibitors, we sought to identify inhibitors of 3CL. 19 SH ESGGG-NH2 + HOOC-G ↓TEPL - Sortase A - Biotin SH ESGGG-TEPL + HOOC-G Figure 7: Self-ligation of biotin-LPETG substrates onto N-SrtA. The substrate (GGGSE)3 was fused to the N-terminus of SrtA (N-SrtA). Upon incubation with the LPETG substrate conjugated to biotin, SrtA is able to self-ligate the biotin substrate onto itself. SH LPET↓G-COOH + H 2N-G2 SH LPET-G5 + HOOC-G T7 phage -TMR - Sortase A Figure 8: Self ligation of G2-TMR substrates onto the C-SrtA displayed on the T7 phage. C-SrtA was displayed on the surface of T7 phage. Upon incubation with the di-glycine substrate conjugated to the fluorescent dye TMR, SrtA on phage is able to self-ligate the fluorescent moiety onto itself, thus labeling the phage. 20 It was decided to mutate the active site of the enzyme 3CL and select for good binders from a commercially available peptide phage display library. While incubation with the active enzyme will cleave most of the binders, incubation with the mutant will enable isolation of binders. Upon emergence of a strong binder a group of similar peptides may then be designed, synthesized and the inhibition of the protease can be studied in solution. In this report, a commercially available 7 amino acid peptide library on the phage was used to screen against the active site mutant of the 3CL protease in efforts to identify good peptide binders to the enzyme (see Figure 3). All assay procedures was optimized using Streptavidin as a model protein. Using the affinity selection method, multiple rounds of the library selection were carried out. This was followed by a binding assay to select for good binders. Several low affinity binders were identified and these were characterized by DNA sequencing. 21 2. MATERIALS AND METHODS 2.1 Making chemically competent bacteria for transformation The desired bacterial strain was grown until OD600 reached 0.5 and chilled on ice for 15 min. The cells were harvested at 1681g, 4 °C, for 10 min. 0.5 volumes (of the starting volume of culture) buffer A was added and the pellet was resuspended by pipetting up and down. After 15 min of incubation on ice, the cells were harvested again as above. The pellet was resuspended in 0.04 volumes (of the starting volume of culture) of buffer B, incubated on ice for 15 min. The cells were then aliquoted into 100 µL aliquots, frozen by placing in liquid nitrogen, and placed immediately at 80°C. Competency in the orders of 107/µg of plasmid DNA was obtained using this protocol. For all buffer compositions see appendix A. 2.2 Transformation of plasmids/ligated vectors into chemically competent cells The competent cells were thawed on ice. The DNA (plasmid/ligated vector) was added into the competent cells (the volume of the DNA sample did not exceed 5 % of the volume of the competent cells). Typically 100 µl of competent cells was used per transformation reaction. The tube was gently tapped to allow mixing of the DNA with the cells. This mixture was then incubated for 30 min on ice. A heat shock at 42 °C was given to the cells, for 45 sec. LB media was added (the volume of the mixture was topped up to 500µL with LB) and the cells then incubated at 37 °C, 250 rpm, for 1 hr (for the recovery of the cells from the heat shock and expression of the antibiotic resistance genes). Following this, the cells were either split and plated (100 µl and 400 µl) or all 500 µl was plated, based on the number of colonies expected, on the 22 appropriate LB/antibiotic plates, left to grow O/N at 37 °C until colonies were visible. 2.3 Transformation of yeast cells The yeast strain InvSC1 (INVITROGEN) was used to make competent cells using the S.c. EasyComp. Kit™ obtained from Invitrogen. The preparation of the competent cells and transformation was done according to the company protocol. 2.4 PCR All PCR’s in this thesis, unless otherwise stated, contained the following, in the PCR master mix: 0.2 mM dNTP mixture, 10-50 ng of template DNA, 0.1 µM of each primer and 2.5 U DNA polymerase* in the corresponding polymerase buffer. The PCR program consisted of the following, 15 min , 95 °C; 29 cycles of 30 sec, 95 °C, X ŧ sec, X° ŧ C, X ŧ min, 72 °C; with a final 10 min 72 °C extension. For primers used in various cloning experiments please refer to Cloning Table, Appendix B. *Taq polymerase (Promega) was used for screening (e.g. colony PCR) and optimizing PCR conditions. *Hot Star Taq polymerase (QIAGEN) was used when cloning was intended. *Pwo polymerase (Roche) was used when full length amplification of plasmid DNA was desired. ŧAnnealing temperature was typically set 5 °C less than the lowest Tm of the two primers; ŧannealing time varied in between 30 sec to 1 min, and ŧ time of extension was 1kB/min for Taq polymerase. All PCRs were performed in the PTC-225, Peltier gradient thermal cycler (MJ research). 23 2.5 Cloning 2.5.1 TA cloning The pCR®2.1-TOPO® ( Invitrogen) vector was used for all TA cloning procedures. After PCR amplification of the desired gene, an agarose gel was run to check the yields. If the yield of the PCR product was acceptable (20-40 ng/µL) a 1/3rd reaction volume of that recommended by the company protocol was set up and found to be sufficient to give significant number of colonies. Typically 1.33 µL of the PCR product was combined with 0.33 µL of the salt solution and 0.33 µL of the TOPO vector, incubated at RT for 30 min. Following incubation the entire reaction mix was transformed into chemically competent TOP10 cells (Invitrogen) and plated onto XGal/LB/Amp plates. Blue colonies are non recombinants and the white ones are recombinants. 2.5.2 Gateway cloning To clone genes into gateway destination vectors, primers were designed that flank the gene of interest, and also carry the necessary recombination sites (AttB) required for the recombination reaction. See Cloning table, Appendix B, for all primers. After the production of the AttB-PCR products, a BP cloning was set up. Normally 1/8th of the reaction volume recommended by the manufacturer (Invitrogen) was found to be sufficient for a BP reaction (see Figure 9). A 1/8th BP reaction typically contained 515 fmol of the attB PCR product, pDONR™ vector (pDONOR201) ~20 ng, 0.5 µL -1 µL of the BP Clonase™ mix, 0.5 µL of the 5 X reaction buffer and TE (10 mM Tris and 1 mM EDTA, pH 8) to 4 µL. The reaction mix was incubated at 25 °C for 12 hrs. At the end 0.25 µl of Proteinase K solution was added and the incubation was 24 Figure 9: The BP cloning reaction. Facilitates recombination of an attB substrate (attB-PCR product or a linearized attB expression clone) with an attP substrate (donor vector) to create an attL-containing entry clone. This reaction is catalyzed by BP Clonase™ enzyme mix. (Figure and caption are taken from Gateway® Technology, catalog number: 12535-019). Figure 10: The LR cloning reaction Facilitates recombination of an attL substrate (entry clone) with an attR substrate (destination vector) to create an attB-containing expression clone. This reaction is catalyzed by LR Clonase™ enzyme mix.(Figure and caption are taken from Gateway® Technology, catalog number: 12535-019). 25 continued at 37 °C. The entire mix was used for transformation into TOP10 cells. BP recombinants were identified using a PCR screen with appropriate primers and sent for DNA sequencing. Upon verification of the sequence, the BP construct was ready for a LR reaction. One fourth of the reaction volume recommended by the manufacturer (Invitrogen) was found to be sufficient for a LR reaction (see Figure 10). A ¼th LR reaction typically contained 75 ng of the entry clone, 75 ng of the destination vector, 0.5 µL of the LR clonase™ mix, 1 µL of the 5X reaction buffer and TE (10 mM Tris and 1 mM EDTA, pH 8) to 5 µL. The reaction mix was incubated at 25 °C for 12 hrs. Following this 0.4 µl of Proteinase K solution was added to it (supplied with the kit) and incubated for 10 min at 37 °C. The entire mix was used for transformation into TOP10 cells. Upon PCR verification of LR recombinants, the LR construct was transformed into the suitable expression host. 2.5.3 RE-based cloning into conventional plasmids and large bacteriophage genomes To clone genes into conventional plasmids, the following procedures were used. The desired genes were PCR amplified (for primers used in the cloning of different genes see cloning Table, Appendix B), cloned into the pCR®2.1-TOPO® and sequence verified. Following this the clone was digested at the designed enzyme sites (enzyme sites were normally added onto the primers, for details see Cloning Table, Appendix B) and gel purified using the QIAquick gel extraction kit™ (Invitrogen). The gene thus prepared was ligated into appropriate vectors (vector was linearized using the same enzymes, purified using the QIAquick gel extraction kit™ (Invitrogen), dephosphorylated using Shrimp Alkaline phosphatase (Promega) and purified again using the QIAquick PCR purification kit™) using the T4 DNA Ligase (Promega). To 26 clone into huge bacteriophage genomes, the procedures for preparation of the insert gene remained the same as above (see Figure 11). The 10-3-B T7 DNA was extracted as per the protocol from the Lambda Mini purification kit (Qiagen). Buffer L1 from the kit contained RNAse and DNAse, which were found to degrade in a few months after the kit was opened. Because of this the extracted DNA was found to contain huge smears when run on the gel to check for purity. Addition of RNAase (0.3 mg/10 mL lysate) and DNAase (90 µg/10 mL lysate) to the clarified lysate before incubation at 37 °C with buffer L1 (Step 1 of the protocol), and also an additional proteinase K treatment (0.2 mg/mL) during step 6, solved this problem. While the digestion of the T7 vector DNA followed conventional restriction enzyme digestion procedures, purification of large fragments of DNA (greater than 10 kB, e.g., the 37 kB, 10-3-B vector from Novagen) using the regular commercial gel/ PCR purification kits was extremely inefficient. Hence several other alternatives were considered. The only purification system that has given the highest yields is Agarase (available from several sellers, NEB, Fermentas etc...) enzyme digestion of the agarose gel (efficient , greater than 90% yields), followed by ethanol precipitation of the DNA. But it was found that packaging (see Section 2.16.1) of the ligated DNA into the T7 packaging extract (Novagen) is severely impaired when the DNA was purified using agarase (presumably the carbohydrate moieties that are left over from digestion of the agarase gel, precipitates during ethanol precipitation and affects the packaging reaction). Yields upto 30% was obtained after gel purification of large DNA using the QiaExII beads from Qiagen. But for regular cloning purposes, when purification was intended, a simple ethanol precipitation of DNA [52] was sufficient. The small DNA fragment resulting from the digestion of the vector will not be precipitated very efficiently, and not interfere with the cloning. Since large DNA fragments are susceptible to shearing 27 from vigorous pipetting, when possible large bore tips and gentle pipetting was used. Separation of large fragments of DNA (the 20 kB and the 17 kB fragment obtained after digestion of 10-3-B) was done by running the fragments in a 0.4 % agarase gel, at 15 V, for greater than 6 hours up to O/N. For further information on the different genes that were cloned and the different vector back bones used please refer to Cloning Table, Appendix B. 2.6 Sequencing of genes All genes were sequenced using the ½ reaction recommended by the ABI prism manual. BIG dye V3.1 (ABI), was used and the genes were sequenced using the ABI 3100A sequencer. A typical sequencing PCR mix contained the following, 100-150 ng of Template DNA, 4 µL of the BIG dye reaction mix (with polymerase and ddNTPs) 3.2 pmoles of each primer, 2 µL of the 5X reaction buffer in a total of 20 µL reaction volume, and subjected to the following PCR program, 24 cycles of 96 °C, 30 sec; 50 °C, 15 sec; 60 °C, 4 min. 2.7 Site directed mutagenesis of genes Site directed mutagenesis of genes was carried out by designing the mutation (typically one to two base pair substitutions) into a forward primer that flanks the mutation by 20-25 base pairs on either side of the mismatch. The reverse primer was the exact complement of the forward primer. PCR of the whole plasmid was then carried out using special long half-life high-fidelity polymerases. The mutations were 28 RE digest of the Sortase A gene PCR amplification of SortaseA from the S.aureus genome Sortase Sortase Sortase Cloning of Sortase A into the pTOPO vector 1 MCS 10B 2 The T7 genome-10-3-B vector 10B MCS Phage heads RE digest of the 10-3-B vector with BamH1 and XmaI 1 Packaging into the T7 phage packaging extract 10B + Ligation of the SortaseA gene into the cut 10-3-B vector 2 Sortase 3 1 Phage tails + 2 3 Packaged T7 phage can be amplified and the recombinants are identified using PCR screening Figure 11: RE-based cloning of SrtA into the T7 phage genome 29 introduced into the template using the primers during the PCR reaction, following which the parental strand without the mutation was digested away. Specific digestion of the parental strand was effected using the enzyme DpnI which cleaves away the methylated strand of the parental DNA (see Figure 12). Either the Pfu Turbo polymerase (Stratagene), or the Pwo polymerase (Roche) was used. When Pfu Turbo polymerase was used, the PCR reaction conditions were similar to the instruction protocol (QuikChange site directed mutagenesis kit from Stratagene). When the whole plasmid template PCR was carried out using the Pwo polymerase (Roche), the PCR reaction mix contained the following. 5 U of the Pwo polymerase, 600 pM of each primer , 0.2 mM each dNTP, 50-100 ng of template, in a 50 µL reaction, using the following program, 94 °C, 2 min; 10 cycles of 94 °C 15 sec; 55 °C, 30 sec; 6 min, 72 °C and 15 cycles of 94 °C, 15 sec; 55 °C, 30 sec; 6 min + 20 sec/Cycle (cycle extension) 72 °C, with a final 10 min, 72 °C extension. Following PCR the parental template was digested using DpnI (NEB) and transformed into XL1blue cells. The transformants were DNA sequence verified. For further information on the different genes that were mutated and the different primers used please refer to Cloning table, Appendix B. 2.8 Expression of different fusion proteins from different vectors and hosts. For details of expression conditions for each Clone/Host pair, see Expression table, Appendix B. All bacterial hosts were grown in LB media prior to and during induction of protein expression. Following expression of proteins, the bacterial cells were harvested at 1681g, 4 °C, for 15 min, and the pellets were frozen at -20 °C. Details of expression of genes from yeast are as follows. Yeast cells (InvSC1, Invitrogen) harboring the clones were maintained in SD-URA + 2 % glucose media 30 Figure 12: Overview of the site directed mutagenesis method. Figure adapted from Quikchange® Site-Directed Mutagenesis Kit, Instruction Manual (Catalog number 200518). 31 (see appendix for composition). An overnight culture of yeast cells (5 mL) was grown and the OD600 was measured. A volume of cells needed to give OD600 = 0.4 in 5 mL of SD-URA + 2 % galactose (Induction media) media was calculated and the same was added to the induction media. The cells were grown for another 24 hrs and samples were removed at various time intervals (2, 4, 6, 12, 24 hrs), to check for expression levels and frozen O/N at -20 °C. 2.9 Western blot of proteins Western blot protocol that used penta-HIS antibody for detection of 6XHIS-tagged proteins is given below. Proteins (pure protein/crude cell lysate) to be detected using a penta-HIS-HRP conjugated antibody, were denatured and run on a SDS –PAGE gel. Transfer of the proteins from the gel to a solid support (PVDF, Amersham) was done using either semi dry transfer method (HOEFER TE 70, Amersham) when the proteins where small (up to 50 kDa) and using the wet transfer method (Mini trans Blot electrophoretic transfer unit, Biorad) when the proteins were larger than 50 kDa. The transfer was done following manufacturers instructions. Following transfer, the membrane was blocked using 5 % skimmed milk dissolved in PBST (Tween 20, 0.1%) either O/N at 4 °C or for 1 hr at RT. Penta HIS HRP conjugate antibody (QIAGEN) was then added at a 1 in 1000 dilution in 5 % skimmed milk dissolved in PBST (Tween 20, 0.1%) to the membrane and incubated for 1 hr at RT. Milk is known to reduce the sensitivity of the anti-HIS antibody (penta-HIS-HRP-conjugate, QIAGEN), and was used only when substantial amounts of protein was expected to be transferred onto the blot. Following this the membrane was washed 6 times, with buffer changes after every 10 min in PBST (Tween 20, 0.1 %) using a suitable shaker. The HRP signal was then visualized using ECL plus (Amersham pharmacia biotech) 32 or Super Signal® west Pico/Dura substrate (Pierce), depending on the amounts of protein expected to be transferred. Likewise for more details on dilution ranges, incubation time, wash conditions of other antibodies used to detect other affinity tags please refer to Expression table, Appendix B. 2.10 Affinity chromatography of proteins 2.10.1 Ni-NTA column The 6X-HIS tagged proteins were purified using Ni-NTA resin (QIAGEN). The frozen pellets (see Section 2.8) were thawed on ice, lysis buffer H1 (for all buffers see Appendix A) was added followed by addition of Lysozyme (Sigma) at 1 mg/mL and incubation was continued on ice for 30 min. The volume of lysis buffer H1 added depended on the expression level of the protein. If the expected yields of protein were less the 1 mg/liter of culture then a 100X concentration was used, e.g., 50 mL starting culture volume was concentrated to 0.5 mL lysis buffer. The cells were sonicated for further lysis. Sonication was carried out with 30% amplitude, 10 sec ON, 10 sec OFF, 12 times, using the SONICS™ ( Newtown, CT, USA) VIBRA CELL. Successful lysis of cells was followed by using a Bio-Rad-Protein assay (BIORAD) which was carried out according to manufacturer’s instructions. The micro-assay procedure was used. The lysed cells were then centrifuged at 20,598g for 30 min at 4 °C. The supernatant was incubated with 100 µL Ni-NTA™ beads / 100 mL culture, for 1 hr at 4 °C. The 6XHIS tagged proteins were separated from other unbound proteins by washing at first with 10 bed volumes of wash buffer H2, followed by wash with 10 bed volumes wash buffer H3. Elution of the 6XHIS tagged proteins was done by the addition of elution buffer H4. Elution of the proteins was monitored using the Bio-Rad protein 33 assay. The fractions containing substantial amounts of proteins were pooled and napped using the NAP™ columns (Pharmacia Biotech) into buffer H5, aliquoted into “use and throw” volumes and frozen at -20 °C. Proteins were napped according to manufacturer’s instructions, using the NAP™ -5 columns. 2.10.2 GSH column The GST tagged proteins were purified using GSH resin (Amersham). The frozen pellets (see Section 2.8) were thawed on ice, lysis buffer G1 (for all buffers see Appendix A) was added followed by the addition of Lysozyme (Sigma) at 1 mg/mL and incubation was continued on ice for 30 min. A 20X concentration was performed, e.g. 200 mL starting culture volume is concentrated to 10 mL lysis buffer. The cells were sonicated for further lysis. Sonication was carried out with 30 % amplitude, 10 sec ON, 10 sec OFF, 12 times, using the SONICS™ ( Newtown, CT, USA) VIBRA CELL. Successful lysis of cells was followed by using a Bio-RadProtein assay (BIORAD) which was carried out according to manufacturer’s instructions. The micro-assay procedure was used in all cases. The lysed cells were then centrifuged at 20,598g for 30 min at 4 °C. The supernatant was incubated with 500 µL GSH beads / 100 mL culture, for 30 min at RT. The GST tagged proteins were separated from other unbound proteins by washing at with 10 bed volumes of wash buffer G2. Elution of the GST tagged proteins was done by the addition of elution buffer G3. Elution of the proteins was monitored using the Bio-Rad protein assay. The fractions containing substantial amounts of proteins were pooled and napped using the NAP™ columns (Pharmacia Biotech) into buffer G4, aliquoted into “use and throw volumes” and frozen at -20 °C. Proteins were napped according to manufacturer’s instructions, using the NAP™ -5 columns. 34 2.10.3 Chitin columns The CBD (chitin binding domain) tagged proteins were purified using Chitin beads (NEB). After expression of proteins in yeast (5 ml), the cells were harvested and frozen O/N at -20 °C. The cells were allowed to thaw on ice and lysis buffer C1, 0.5 ml, was added (a 10X concentration was used). The cells were sonicated for lysis. 30 % amplitude, 30 sec ON, 30 sec OFF, a total of 7 min ON and SONICS™ (Newtown, CT, USA) VIBRA CELL was used. Following this the lysate was checked for fluorescence using the Typhoon 9200 (Amersham -Biosciences) at the EGFP fluorescence wavelength. The lysate was spun down at 20,598g for 30 min at 4 °C. The supernatant was incubated with 25 µL Chitin beads / 5 mL culture, for 30 min at RT. The CBD tagged proteins were separated from other unbound proteins by washing with 10 bed volumes of wash buffer C2. Purification was checked by boiling the beads and running them on a SDS-PAGE gel. 2.11 Production of N-terminal cysteine proteins The cells harboring the respective plasmids were grown in 1 L of LB medium containing 100 mg/L ampicillin at 37 °C in a 250 rpm air shaker. Protein expression was induced at OD600 ~ 0.6 with 0.5 mM IPTG and left shaking O/N at RT. Cells were harvested by centrifugation (5000g, 30 min, 4 °C), resuspended in lysis buffer (20 mM Tris-HCL pH 8.5, 500 mM NaCL, 1 mM EDTA) and lysed by sonication on ice. The cell debris was pelleted down by centrifugation (4000g, 30 min, 4 °C) to give a clear lysate containing the intein-fusion proteins. A column packed with 10 mL of chitin beads was pre-equilibrated with column buffer (20 mM Tris-HCL pH 8.5, 500 mM NaCL, 1 mM EDTA). The clear lysate was loaded onto the column at a flow rate of 0.5 mL/min and washed with 10 volumes of column buffer. The column 35 was then flushed quickly with one column volume of cleavage buffer (20 mM TrisHCL pH 7.0, 500 mM NaCL, 1 mM EDTA) before stopping the flow. The above procedures were carried out at 4 °C to prevent premature on-column cleavage of intein-tag. On-column incubation with the cleavage buffer took place for 20 hr at RT with gentle agitation and the protein was eluted in 2 mL fractions. 2.12 Spotting of N-terminal cysteine proteins on thioester slides Thioester slides were prepared as follows. The epoxy-derivatized slides (see Section 2.14) were incubated with 10 mM diamine-PEG for 30 min. The slides were then washed with deionized water and placed in a solution of 180 mM succinic anhydride for 30 min. This was followed by boiling the slides in water for 2 min. NHS solution was prepared and the slides were incubated with it for 3 hr. Following incubation the slides were rinsed with deionized water and allowed to react O/N with a solution of benzylmercaptan. Finally, the slides were washed with deionized water and dried. 10 µL of the clarified cell lysate (see Section 2.11) was added on to a source plate. The N-terminal cysteine-containing proteins were adjusted to a stock concentration of 1 mg/mL (with PBS buffer, pH 7.4). Stocks with series dilutions were also prepared. The protein was spotted onto the thioester-functionalized glass slides using an ESI SMA™ arrayer. All slides were incubated for 3 hrs and subsequently washed with water for 20 min, followed by detection or storage at 4 ºC. For detection of protein immobilization, slides were scanned with an ArrayWoRx™ either directly under FITC channel, or hybridized with Cy5-labeled anti-EGFP for 1 h and analyzed. 36 2.13 In vitro biotinylation of proteins Following pull down of CBD tagged fusions onto the Chitin column (see Section 2.10.3), biotinylation of the protein was done by quickly flushing the column with buffer C3 and incubating the dry beads for 24 hrs at 4 °C. After the 24 hr incubation the protein was eluted in buffer G2. Following elution the proteins were either checked for the incorporation of the biotin tag using a western blot, or were used for spotting onto microarrays. 2.14 Spotting biotinylated proteins onto avidin slides The slides were cleaned in piranha solution (H2SO4:H2O2, 7:3) for at least 2 hr. Following that slides were washed copiously with de-ionized water and rinsed with 95% ethanol and dried. Then the slides were soaked in 1% solution of Silane (95% ethanol, 16 mM acetic acid) for 1 hr. The epoxy derivatized slides were then washed three times in 95 % ethanol and cured for 2 hrs at 150 °C. The slides were once again rinsed with ethanol and dried. Following this 40-60 µL of 1 mg/ml Avidin (SIGMA) in 10 mM sodium bicarbonate buffer was applied to the slides, covered with a cover slip, and incubated for 30 min. The slides were dried after washing with water. The unbound epoxides were reacted with 2 mM aspartic acid in 0.5 M sodium bicarbonate, pH 9, and covered with a coverslip. Following this the slides were washed with water and dried. The spotting was done manually, 2 µL spots of the samples were applied to the slides and allowed to react for 2-3 hrs in moisture chambers before drying them in air. The slides were then washed by incubating them in PBST (Tween 20, 0.1%) 10 times, 1 min each. Following this the antibody solution was applied to the slide (when required), was covered with a cover slip, allowed to incubate for 1 hr, before washing off the unbound antibody and dried in air.. The 37 slides were then scanned using the ArrayWoRx™ Microarray scanner (Applied Precision) at the respective wavelengths. 2.15 Enzyme activity assays 2.15.1 Testing activity of SrtA Fluorescence assay: SrtA/N-SrtA/N-SrtA-C184A/C-SrtA/C-SrtA-C184A, 20 µM, was incubated with EGFP-LPETG-V5, 20 µM and GG-TMR, 500 µM in reaction buffer R1 to a final volume of 100 µL at 37 °C for 1 hr. The reaction was stopped by adding buffer R2 to the reaction mix and boiling for 10 min at 95 °C. The reaction contents were then separated in a SDS-PAGE gel. Fluorescence of GG-TMR was scanned in gel using Typhoon 9200 (Amersham Biosciences), at the appropriate wavelength. Hydrolysis/transpeptidation activity: To test the hydrolysis activity, SrtA/C-SrtA/CSrtA-C184A, 5 µM, was incubated with ACC-LPETG-DABCYL, 100 µM in reaction buffer R1 to a final reaction volume of 100 µL (see Figure 4). The increase in fluorescence that would be observed due to hydrolysis (the fluorescent moiety and its quencher are separated) was followed using the Spectra Max Gemini XS (Molecular devices). For transpeptidation activity SrtA/C-srtA/C-SrtA-C184A, 5 µM, was incubated with ACC-LPETG-DABCYL, 100 µM and Gly6-His6, 100 µM in reaction buffer R1 in a final reaction volume of 100 µL (see Figure 4). The increase in fluorescence that would be observed due to transpeptidation (the fluorescent moiety and its quencher are separated) was followed using the SpectraMax GeminiXS (Molecular devices). 38 2.15.2 In vitro self-ligation assay To detect self labeling, N-SrtA/N-SrtA-C184A, was incubated with 500 µM, BIOTIN- LPETG, whereas as C-srtA/C-SrtA-C184A, was incubated with 500 µM G5-BIOTIN, in buffer R1 for 1 hr at 37 °C (see Figures 6 and 7). The reaction was stopped by adding buffer R2 to the reaction mix and boiled for 10 min at 95 °C. The reaction contents were then separated in a SDS-PAGE gel. Biotin was detected using anti-BIOTIN antibody; see Section 2.9 and expression table, Appendix B, for more details on the western blot. 2.15.3 Self-ligation assay on the phage SrtA on phage was tested as follows, a 10 mL phage lysate was PEG precipitated (see Section 2.16.3) and re-suspended in 1 mL of buffer R1. 10 µL of this solution was used in a 50 µL reaction volume, using the same conditions as in Section 2.15.2 (see Figure 8). Reaction aliquots were taken at required time intervals and the reaction stopped by the addition of buffer R2, followed by boiling at 95 °C for 10 min. Fluorescence tagging of the proteins was detected after running a SDS-PAGE and scanning the gel using the Typhoon 9200 (Amersham Biosciences). 39 2.16 General phage methods 2.16.1 Packaging of T7 phage DNA One tube of the T7 DNA packaging extract (Novagen) was thawed on ice and aliquoted into 4 tubes with 6.25 µL each. To this 1.25 µL of the ligation mix was added and stirred gently with a pipette tip. The reaction was allowed to proceed at RT for 2 hrs and was stopped by the addition of 75 µL of LB. The packaged phage was plated out (see Section 2.16.4) or stored up to 24 hrs at 4 °C. If long term storage is intended then phage was propagated and glycerol stocks were made. 2.16.2 Amplification of phages M13 phages: The host cells (ER 2738, NEB) were grown to an OD600 of 0.095 at 37 °C in LB broth supplemented with ampicillin at 100 µg/mL. The eluted phages were then added to the culture and allowed to grow until 4 hrs at 37 °C. The number of host cells was always in excess to the number of phages. All eluents were amplified in this manner. To amplify single plaques, plaques were picked into 1 mL OD600 = 0.095 host cells and allowed to grow for 4 hrs at 37 °C. Following amplification the bacterial cells were pelleted at 10,000g for 10 min at 4 °C. The supernatant contained the phages. Rescue of phagemid: The XL1 blue cells containing the appropriate phagemid clones were grown at 30 °C in 2XYT broth supplemented with 2 % glucose (Glucose is added to the growth media to repress the protein expression from the Lac promoter of the phagemid prior to the addition of helper phages, overexpression of Gene III is toxic to the bacterial cells) and ampicillin, 100 µg/ml until a OD600 of 0.5. The cells 40 were then harvested and re-suspended in 2XYT broth (see Appendix A) containing ampicillin, 100 µg/ml, Kanamycin, 50 µg/ml, helper phages (M13-K07, NEB) at MOI 0.1 and IPTG 0.4 mM. The incubation was continued at 37 °C for 1 hr without shaking, followed by incubation at 30 °C O/N at 250 rpm. Upon removal of cells the supernatant contained the phages. T7 phages: The host cells (e.g., BLT5403, Novagen) were grown to an OD600 of 0.5 – 0.8 at 37 °C in LB broth supplemented with ampicillin at 50 µg/mL. Phages were then added at a MOI of 0.001–0.01 (i.e., 100–1000 cells for each pfu) and allowed to grow until lysis was observed, typically 2 to 3 hrs at 37 °C. Following this the bacterial cell debris was pelleted at 10,000g for 10 min. The supernatant contained the phages. Amplification of single plaques was also done in a similar manner. 2.16.3 PEG precipitation of phages. PEG precipitation of the phages was done by the addition 1/6th volume of buffer C (M13 phage) or 1/6th volume of buffer D (T7 phage) to the supernatant (see Section 2.16.2), followed incubation on ice for a minimum of 1 hr up to O/N. Centrifugation was done at 10,000 g, for 15 min at 4 °C for 10 min to pellet the phages. The phages thus obtained were re-suspended in the desired buffer and desired volume. 2.16.4 Storage, Serial dilution and titering of phages. Phage (M13/T7) is the most stable in LB and could be stored for several months in LB, at 4 °C, without any loss of titers. For long term storages however glycerol stocks were be made (15 % glycerol was added to the phage supernatant and frozen at -80 °C). Serial dilution of phages was done in LB. Typically 100-fold dilutions, were 41 done in 96-well plates, using a 8-channel pipette, in a 96-well plate shaker, to shake in between dilutions. Up to 8 different samples were processed simultaneously, using just 8 tips (there was no need to change tips in between dilutions, just pipetting slowly was enough to prevent cross contamination). Titers from 96-well plates were counted on appropriate LB-Host-bacterial plates (The host bacteria (250 µL/90 mm dish), was grown to an OD600 ≥1, mixed with melted top agar (7 mL/90 mm dish) , at 55 °C, poured onto to LB/Antibiotic plates and allowed to solidify to make host bacterial plates; the procedure is the same for both M13 and T7 phages) using multi channel pipettes and spotting 2 µL volumes from each sample. Upon the appearance of plaques (8 hours, 37 °C for M13 phages, 3 hrs 37 °C for T7 phages) spotted areas containing discrete phages were counted and multiplied by the dilution factor to obtain the actual number of phages. All titers were done in triplicate and the average values were used. Typical phage titers for M13 phage after amplification was 1012 pfu/mL , for T7 phage was 1010 pfu/mL , eluted titers varied between 0-105, serial dilutions were done accordingly. 2.16.5 Plaque lift T7 plaques can be lifted onto nitrocellulose membrane and probed for protein expression using antibody. The plaques (500- 750 in number) were mixed with host bacteria (250 µl of OD600 ≥ 1 host bacteria /90 mm dish) and 7 mL of top agar/90 mm dish and plated out on LB/Amp plates. Once the plaques grow to 2-3 mm sizes the plates were chilled at 4 °C for a minimum of 1 hr. Nitrocellulose membrane cut to the size of the Petri-dish was marked on one side with a pencil, and laid on the agar plate gently. After a few min, the membrane was lifted out gently and allowed to dry for at 42 least 20 min. Following this the membrane was processed like a regular western blot membrane for any antibody. 2.16.6 Sequencing phage 2.16.6.1 M13 The plaque whose DNA was to be sequenced was amplified in a small volume of 1 mL. After amplification the cells were pelleted and 500 µL of the supernatant was removed into a fresh tube. Following this 200 µL of buffer C was added to the supernatant, mixed, and let to stand at RT for 10 min. The sample was then centrifuged at 10,000g for 10 min. The phages were pelleted at this step, and the supernatant was discarded. The pellet was re-suspended in 100 µL buffer E and 250 µL ethanol, allowed to stand for 10 min. The DNA was recovered by spinning for another 10 min at 10,000g. The pellet was washed with 70 % ethanol, and air dried. The pellet was then re-suspended in 10 µL of TE buffer (10 mM Tris-HCL (pH 8.0), 1 mM EDTA), and 5 µL was used for DNA sequencing (See section 2.6). 2.16.6.2 T7 Genes to be sequenced were amplified using the T7 up and T7 down primers (see Cloning table, Appendix B), purified using QIAGEN PCR purification kit, and used as templates for sequencing with the same primers. See Section 2.6 for DNA sequencing. 43 2.17.7 Phage enrichment methods 2.17.7.1 Affinity based enrichment of C-SrtA-T7 Phages bearing the SrtA protein were diluted at 1 in 104 or 1 in 105 of bare T7 phages (wildtype T7). This pool of phages was then incubated with 25 µL of affinity beads (Ni-NTA beads were washed in buffer F and blocked for 1 hr at RT in blocking buffer H), in the binding buffer F and incubated for 1 hr at 4 °C in a rotating platform. Following incubation the unbound phages were washed away using 0.1% PBST, 1 min each, 5 times. The bound phages were eluted by infecting the beads with 1 mL OD600 = 0.5 BLT5403 cells, and incubated until lysis occurred at 37 °C. The lysate was then centrifuged at 10,000g for 10 min to remove the bacterial debris. 100 µL of the supernatant was used directly for the next round of affinity enrichment. 2 µl of the phage supernatant was boiled in 100 µL of water for 10 min and 2 µL of this solution was used as template in a PCR reaction to detect enrichment using T7 Up and T7 down primers. The selection was continued for 6 rounds. 2.17.7.2 Activity based enrichment of C-SrtA-T7 Activity based enrichment was carried out essentially in the same way as affinity based enrichment with the following modifications. Streptavidin beads (Promega)/Streptavidin microtiter plates (PIERCE) were washed with wash buffer G2 and blocked with buffer H, incubated with 0.5 mM or with 10 µM of G5-BIOTIN respectively at RT for 30 min. The unbound G5-BIOTIN was removed by washing with 0.1% PBST at least 3 times. The phage bearing the C-SrtA was diluted at 1 in 102 to 103 of bare T7 phages (wildtype T7), and incubated with the beads/plate in buffer R1 at 37 °C for 2 hrs. 13 rounds of enrichment were done. The following was 44 also tried, 0.5 mM of G5-BIOTIN was incubated with the diluted phages (see Section 2.17.1.1) in buffer R1 for 2 hrs at 37 °C, the un reacted G5-BIOTIN was removed by PEG precipitation, and the phages were incubated with SA beads (washed with wash buffer G2 and blocked with buffer H) for 30 min at RT. The unbound phages were washed away and the bound were eluted as in Section 2.17.7.1. 2.17.7.3 Biopanning against SA and 3CL mutant. Biopanning against the Streptavidin (SA) model protein and the 3CL mutant protease was done as follows. SA coated plates (High Binding capacity SA plates, PIERCE) was used for panning against SA, and 25 µL of GSH beads was used to pan against the GST fusion protein, 3CL-C145A-GST (10 nM concentration of the 3CL-C145AGST was used per assay ). The SA plates or the 3CL mutant bound beads (washed in PBS and blocked using block buffer H) were incubated with 2 x 1011 phage in 100 µL final volume of PBS for 1 hr at RT. Following incubation the unbound proteins were removed by washing 10 times, 1 min each with 100 µL of PBST (Tween 20, 0.1%). Elution of the bound phages was done using 100 µL of buffer G for 15 min at RT. This was neutralized immediately using 150 µL of 1M Tris, pH 9.1. A small amount of the eluent (10 µL) was used for titering and the rest was amplified as described above. The subsequent rounds were performed using 2 x 1011 phages from the amplified eluent. A total of 3 rounds were performed, at the end of which isolated phage plaques were sent for DNA sequencing to determine the peptide sequence. 45 2.17.7.4 Binding assay For each clone/target protein to be tested, 1 µg of the target protein was immobilized in separate wells of ELISA microtiter plate (NUNC). Immobilization of target was done by allowing the target to react with the ELISA plate in 100 µL of PBS buffer (see buffer G2, appendix A), pH 7.4, O/N at 4 °C. The unbound protein was washed off by rinsing the wells 3 times with PBST. The individual phage clones (109 pfu/each of cleared phage lysate) were added to the wells and allowed to react for 1-3 hrs at RT. The unbound phages were washed off using 5 washes, 1 min each with 100 µL of PBST (Tween 20, 0.1%). Elution of the phages was done using 150 µL of 200 mM glycine-HCL (pH 2), for 15 min at RT. The phages were then transferred to a new plate containing 150 µL of 1 M NaPO4 buffer (pH 7.5). The output was then titered, and the percentage of input pfu recovered from the binding reaction was calculated. 46 3. Results and Discussion In this thesis three different approaches to protein engineering and modulation have been evaluated. The first involves an intein mediated method to site specifically engineer proteins for immobilization onto the microarray. The second one is a selection scheme that should enable protein evolution of an enzyme SrtA has been designed and setup. As a third approach to protein modulation a phage display library of random peptides was used to identify binders for a viral protease. The identified binders may be used to design good inhibitors against the enzyme. The results and discussions from these three different projects will be presented separately in Sections 3A, 3B and 3C. 47 3A The intein mediated approach to site-specifically label proteins. An enzyme-mediated approach to modify proteins site-specifically is presented in this section. Two different approaches are described. The first one enables generation of N-terminal cysteine proteins, while the second allows biotinylation at C-terminus of proteins. 3A.1 The intein based method to produce N-terminal cysteine proteins The Ssp Intein tag was used to generate N-terminal cysteine containing proteins for site-specific immobilization onto thioester functionalized glass slides by means of a highly specific chemical reaction known as native chemical ligation [109]. Terminal cysteine containing proteins were generated using the pTWIN vectors. These vectors allow the expression of target proteins with the self-cleavable modified Ssp DnaB Intein having a chitin binding domain fused at their N-termini. The recombinant protein was engineered by standard PCR-based methods and subsequently expressed to have a cysteine residue at its N-terminus by inducing intein splicing at pH 7. The N-terminal cysteine-containing protein thus produced was site-specifically immobilized onto thioester-functionalized slides via the chemoselective native chemical ligation reaction [6]. Only the terminal cysteine residue reacts with the thioester to form a stable peptide bond; other reactive side chains, including internal cysteines, do not react to form a stable product. 48 3A.1.1 Expression of N-terminal cysteine-containing proteins from bacteria. EGFP was cloned into the pTWIN vector to allow it to be expressed as a fusion to the Ssp DNAB intein at its N-terminus. An extra cysteine residue was introduced at the junction of EGFP and Intein. The construct thus obtained was sequence verified and transformed into ER2566 host cells for expression. In order to obtain a pure Nterminal cysteine-containing protein, it was necessary that a sufficient amount of the uncleaved CBD-intein fusion protein was obtained before binding to a chitin column. By performing the protein expression at room temperature for 12 hrs, we were able to obtain a substantial amount of the fusion (at least 50 %; Lane 1 in Figure 13(a)) before chitin column purification. In vitro cleavage of the fusion protein was further reduced by carrying out on-column loading and washings at 4 °C. On-column cleavage and purification conditions were also optimized by carrying out the oncolumn cleavage reaction at room temperature for 12 hrs using the cleavage buffer (pH 7.0) (see Section 2.11). On average, highly purified N-terminal cysteine- containing EGFP fractions could be routinely obtained with sufficient yield (~ 1.5 mg/mL; Lanes 2-4 in Figure 13(a)), and directly used for spotting onto thioester slide. 3A.1.2 Spotting N-terminal cysteine-containing EGFP onto thioester slides To confirm that the N-terminal cysteine-containing EGFP obtained this way is suitable for protein microarray generation, the concentration of the protein sample was adjusted to 1 mg/mL, and other series dilutions, before spotting onto a thioesterfunctionalized glass slide. Successful immobilization of EGFP was unambiguously confirmed by probing the slide with a Cy5-labeled anti-EGFP antibody (see Figure 13(b)). The spots arrayed on the glass slides were generally clear and defined and with relatively low background signals. As observed from the native fluorescence of 49 the immobilized EGFP (Figure 13(c)), relative spot intensity reached saturation at about 0.2-1 mg/mL, indicated the upper limit of the amount of a protein that may be immobilized with this strategy. Significantly, as little as 0.005-0.01 mg/mL of EGFP was sufficient to give an easily detectable signal. The observation of native EGFP fluorescence also served to confirm the proper folding of EGFP upon immobilization. 3A.2 The intein mediated method to site-specifically label proteins derived from yeast The intein mediated strategy for C-terminal biotinylation of proteins and spotting onto to a microarray has been previously explored in our lab [7-11]. In this project the feasibility of producing proteins in yeast, biotinylating them at the C-terminus using the intein mediated method (see Figure 2) and immobilizing them onto microarrays in a high throughput fashion was evaluated. Bacterial, mammalian and cell free systems have been previously used to express the target proteins prior to biotinylation either in vitro or in vivo [8-11]. Bacterial systems while providing good levels of expression, lack the complex environment present in the eukaryotic cell, e.g., they do not have post translational modifications of proteins. While it is feasible to express proteins in the mammalian system it is expensive and the yields are quite low. So the yeast was considered as an alternative. Yeast genetics is very well established, the yields of protein is acceptable and culturing yeasts is inexpensive. It was envisaged that if a high throughput system of expressing and biotinylating proteins from yeast could be setup, then the entire array of enzymes from yeast could be immobilized onto avidin surfaces and studied in a parallel fashion. While the 6000 yeast proteins have been immobilized in the microarray [110], functions of many ORF’s still remain to be discovered, an enzyme array typically consisting of all known and putative enzyme 50 a) Lane No 1 kDa 2 3 4 5 175 67 45 Fusion 27 EGFP Intein b) 70 50 30 10 -10 -100 100 300 500 700 900 1100 EGFP Conc . ( mg/ mL) c) Figure 13: Results from N terminal immobilization strategy (a) Expression and purification of N-terminal cysteine-containing EGFP by in vitro intein-mediated cleavage on a chitin affinity column. Lane 1: Cell lysate containing overexpressed CBD-intein-EGFP fusion protein (~50 kDa). Both the fusion and in vivo-cleaved EGFP (i.e. the 27 kDa band) were observed. Lanes 2-4: The first 3 fractions eluted from chitin column. Lane 5: Proteins retained in the chitin column after elution. (b) Immobilization and detection of purified N-terminal cysteine-containing EGFP on a thioester slide. Varied concentrations of EGFP (left to right in mg/mL: 1, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005) were spotted, in triplicate, and probed with Cy5-labeled anti-EGFP antibody. The native fluorescence of immobilized EGFP was also measured and plotted graphically in (c). 51 ORF’s would facilitate such a process. Also we sought to find out the most suitable intein (from the three commercially available inteins [31]) for use with the yeast system, because it is known that the expression and cleavage pattern of different intein fusion in different expression systems can be quite different [6-10]. Accordingly the C-terminal biotinylation system previously tested out in the bacterial and mammalian cells were extended onto yeast. 3A.2.1 Expression levels and the in vivo cleavage pattern of the Intein-fusion proteins in yeast The genes EGFP-Intein 1 (Intein1: Mxe GYR Intein from pTWIN1 vector, NEB), EGFP-Intein 2 (Intein 2: Mth RIR Intein from pTWIN2 vector, NEB), and EGFPIntein 3 (Intein 3: Sce VMA Intein from pTYB1 vector, NEB) were cloned into the gateway system vector pYES-DEST52, using the gateway methodology (see Figures 9 and 10). The pYES-DEST52 vector is meant for expression of proteins from yeasts, the cloned gene is placed under the “GAL” promoter inducible by the addition of galactose to the medium. After DNA sequence verification of the genes the plasmids containing the genes were transformed into yeast INvSC1 strain. The expression levels of the proteins from the yeast host were monitored by scanning for the native fluorescence of EGFP in different time intervals (see Figure 14). As evident from Figure 14, the best expression levels were obtained from constructs that had the Intein-3 fused to the EGFP, followed by that obtained from Intein 1. Intein 3 has its origin from the yeast Saccharomyces cerevisiae, while Inteins 1 and 2 were from bacterial origins. The Intein 2 fusion was not expressed well in the yeast. A western 52 4 1 3 2 6 5 Figure 14: Native fluorescence of EGFP-Intein fusion from yeast after cell lysis and clarification. 1, 4: EGFP-Intein1 fluorescence after 0 and 24 hrs post induction. 2, 5: EGFP-Intein 2 fluorescence after 0 and 24 hrs post induction. 3, 6: EGFP-Intein 1 fluorescence after 0 and 24 hrs post induction. Fluorescence was detected by scanning through using the Typhoon 9200 scanner at Excitation λ: 488nM 100kDa 73kDa 54kDa 50kDa 1 2 3 4 5 6 7 8 Figure 15: Expression time line of the three EGFP-Intein-CBD fusions in the yeast host detected using anti-CBD western blot.1: EGFP-Intein2-CBD, after 24 hrs of expression. 2: EGFP-Intein1-CBD, after 24 hrs of expression. 3: EGFP-Intein3-CBD, after 10 hrs of expression. 4: EGFP-Intein2-CBD, after 10 hrs of expression. 5: EGFP-Intein1-CBD, after 10 hrs of expression. 6: EGFP-Intein3, after 4 hrs of expression. 7: EGFP-Intein2, after 4 hrs of expression. 8: EGFP-Intein1, after 4 hrs of expression. 8: EGFP-Intein3, after 24 hrs of expression. Red arrows indicate expression after 24 hours of EGFP-Intein1 and EGFP-Intein3. 53 blot with anti-EGFP antibody corroborated the fluorescence pattern observed with the protein expression pattern observed on gel (see Figure 15). Also for the success of the C-terminal Biotinylation strategy it is important that the fusion is obtained intact so that it can be pulled down using the chitin beads and will be available for biotinylation. From the western blot results (see Figure 16) it was evident that at least 30% of the fusion was cleaved off inside yeast for the constructs with Intein 1 and 3 and almost no fusion was detected in the case of the Intein 2. But however since substantial fusion ~ 70% still remained in the cell for Intein 1 and 3 fusions, and because of the higher levels of expression from the Inteins 1 and 3, it was decided to use only Intein 1 and 3 fusion proteins for all further experiments. 3A.2.2 On-column cleavage and generation of biotinylated protein The fusion protein Intein 1/3 -EGFP was purified on chitin beads from a 5 mL yeast culture, and run on a SDS-PAGE gel. It was found that the levels of expression, and yields after purification were such that only faint bands were visible after the coomasie staining of the gel (see Figure 17). Nevertheless due to the very small amounts of proteins that is required for spotting onto a microarray (fg/spot, [110]) it was decided to proceed further and biotinylate the proteins using cysteine-biotin. Upon incubation of the fusion protein with MESNA (a thiol reagent that induces cleavage of the engineered intein from the fusion) and cysteine–biotin the splicing process of the engineered intein was initiated and a thioester group was generated at the C-terminus of the EGFP. This group was then attacked by the SH group of the cysteine from cysteine – biotin to yield a native peptide bond with biotin on the Cterminus (see Figure 2). Upon cleavage and elution using MESNA and Cys-biotin, 54 1 2 3 100kDa 73kDa 55kDa In vivo cleaved Intein 3 ~ 52kDa 40kDa In vivo cleaved Intein 2 ~ 22kDa In vivo cleaved Intein 1 ~ 28 kDa Figure 16: In vivo cleavage pattern of the three EGFP-Intein fusions in yeast crude cell lysate as detected using anti-CBD western blot.1: EGFP-Intein1-CBD, 2: EGFPIntein 3-CBD, 3: EGFP-intein2-CBD. 1 2 3 EGFP + Intein 3 Fusion Cleaved Intein 3 Figure 17: Purification of EGFP-Intein 3 from yeast small scale cultures. Lane 1: 20 µL of EGFP-Intein-3 bound to chitin beads boiled with buffer R2 (without DTT). Lane 2: Blank. Lane3: 20 µL of EGFP-Intein-3 bound to chitin beads boiled with buffer R2 (with DTT). Note: DTT induces cleavage of intein from the fusion. 1 2 3 4 5 6 25kDa Figure 18: Effect of different concentrations of Cys-Biotin on the biotinylation efficiency of EGFP produced in different hosts. 1, 2: EGFP-biotin cleaved off from EGFP-Intien-1, using 10mM and 5mM Cys-Biotin respectively. 3, 4: EGFP-biotin cleaved off from EGFP-Intien-3, using 10mM and 5mM Cys-Biotin respectively. 5, 6: EGFP-biotin cleaved off from EGFP-Intien-1, using 10mM and 5mM Cys-Biotin respectively. 1-4: Yeast host, 5, 6: Bacterial host. Biotinylation was detected using anti-biotin western blot 55 a western blot (see Figure 18) using anti-biotin antibody revealed a band corresponding to the biotinylated EGFP. The effect of the concentration of cysteinebiotin on the biotinylation efficiency was evaluated, 10 mM cysteine-biotin gave the best levels of biotinylation. Also Intein-3 fusions gave higher yields than Intein-1 fusions (see Figure18). Hence it was decided to choose Intein-3 as the final intein of choice for all further experiments. 3A.2.3 Detection on the microarray Following biotinylation the proteins that were eluted, were napped to remove the excess cysteine–biotin. Then the proteins were spotted manually onto avidin functionalized glass slides, with FITC-biotin as control and probed with anti–EGFPFITC. Upon incubation, washing and scanning for fluorescence using the array scanner the FITC-Biotin controls retained their fluorescence, while no significant fluorescence was found either from native the fluorescence of EGFP or from the anti –EGFP-FITC spots. The levels of protein obtained from a small scale culture of yeast was thus was not high enough to obtain immobilization onto the array. Indeed when biotinylated proteins from bacteria were used for immobilization we were able to see significant native EGFP fluorescence on the slides. Moreover biotinylation efficiency though quite high ~ 70%, considering the levels of protein obtained in the first place may not have been sufficient enough to allow immobilization onto the microarray. Although this was not a problem as it only requires protein purification from large scale cultures, it was realized that purification of hundreds of proteins from yeast in a large scale fashion was tedious and not practical. Hence it was concluded that despite the low levels of proteins needed for microarray immobilization, the expression level of proteins in yeast and the efficiency of the subsequent steps of biotinylation were 56 such that, high throughput production and biotinylation of proteins from yeast was not feasible. 57 3B Designing a selection scheme to evolve SrtA on phage SrtA, a transpeptidase isolated from the Staphylococcus aureus bacterium, has proven useful in protein ligation and purification applications [13, 101]. Here in this project it was hypothesized that SrtA could be used to ligate fluorescent probes to proteins engineered to have the LPETG motif and ultimately be useful for imaging proteins in live cells. SrtA is very robust in living cells [88], but in vitro the performance of SrtA is compensated. Several reasons have been proposed for this. In vitro analysis indicated that a weak km of 5.5 mM was observed for the LPETG substrate, a km of 140 µM was observed for the penta-glycine substrate and a kcat of 0.27/sec was observed for the enzyme [99]. The wild type SrtA and its two natural substrates are membrane anchored. The SrtA DNA sequence that was cloned lacks the membrane anchor to make it soluble. The crystal and solution structures of Sortase were obtained without the membrane anchor and hence the importance of the truncated 59 amino acids is unknown. Calcium binding stimulates SrtA; hence it has been proposed that the full length SrtA might form specific interactions with the membrane phospholipids that would contribute to its activity in vivo [99]. 3B.1 The N-terminus extension scheme To improve the activity of SrtA in vitro it was decided to evolve SrtA using protein evolution techniques. Protein evolution of an enzyme as described before requires a selection scheme that will allow the isolation of the desired enzymes from a pool of random mutants. In this project a selection scheme for the evolution of SrtA was designed and successfully set up. Using this selection scheme and a whole library of random mutants of SrtA, it may be possible to evolve more active members of the SrtA enzyme. The selection scheme (see Figure 5) is similar to the product capture 58 approach to evolve enzymes [81, 82]. Two different selection schemes were set up. One, the N-terminal extension scheme, displays a N-SrtA (N-terminus extended with (GGGSE)3 amino acid sequence) on the phage M13. SrtA was fused to one of its substrates 2HN-GGG on its N-terminal side (see Figure 7). It was hypothesized that if the N-terminus is extended enough it should bring the substrate of SrtA into its own active site. The distance between the active site cysteine 184 and the N-terminus was estimated to be around 26.79 °A. It was estimated that 8-10 amino acids residues should be enough to extend the N-terminus of SrtA to its active site (note that SrtA was cloned in without the original N-terminal 59 amino acid residues to keep it soluble). Accordingly a 15 amino acid peptide sequence GGGSEGGGSEGGGSE was fused to the N-terminus of SrtA. The “SE” repeat was known to confer flexibility to peptides and is generally used as linker sequence [112]. It was foreseen that the Nterminal extended SrtA both on the phage and in solution, would react with Biotin – LPETG-COOH and thus label itself (see Figure 7). Thus if a pool of active and inactive members of SrtA was displayed on the phage each with their N-termini modified, only the active ones will react to label themselves, and they can be isolated using avidin beads, amplified and enriched. The M13 phage display has some inherent disadvantages [113]. The protein to be displayed has to be secreted out into the periplasmic space of the bacterial cell wall. Not all proteins retain activity after being secreted into the periplasm and some resist transport. To circumvent this shortcoming, other bacteriophages like the T7 phage have been used to display proteins [114]. Proteins displayed on the T7 phage need not be secreted as T7 phage is a lytic phage, moreover proteins can be displayed as C terminal fusion to the phage capsid protein 10B. Thus considering the fact that SrtA 59 might resist export or might not be active after export into the periplasmic space when packaged into the M13 phage, we decided to display SrtA on the T7 lytic phage. Also whether N- or C-terminal ligations would work was unknown at that time. 3B.2 The C-terminus extension scheme The C-terminal selection scheme is similar to the N-terminal scheme. It displays a CSrtA (C-terminus extended with LPETG) on the T7 phage. SrtA was fused to its substrate LPETG on its C-terminal side (see Figure 6). It has been previously shown that SrtA fused to the LPETG substrate on its own C-terminus, can catalyze the transfer of a Gn peptide onto itself [101]. Based on this report, it was decided to fuse LPETG to the C-terminus of SrtA and display it on the T7 phage. It was foreseen that the C-terminal extended SrtA both on the phage and in solution, would react with G5Biotin and thus label itself (see Figures 6 and 8). Thus from a pool of active and inactive members of SrtA displayed on the phage each with their C-termini modified, only the active ones will react to label themselves, and they can be isolated using avidin beads, amplified and enriched. 3B.3 Activity of SrtA with N- and C-terminal extensions As a proof of the concept to the selection scheme, it was decided to prove that N-SrtA and C-SrtA would carry out the self-ligation in solution, i.e., not bound to the surface of the phage. Accordingly, N-SrtA/N-SrtA-C184A with N-terminal sequence extension (GGGSE)3, and the C-SrtA/C-SrtA-C184A with C-terminus extension (GGLPETG) were constructed by adding on the required sequences onto the primers, cloned into vectors pFAB5c.HIS-Exp and pDEST17 vectors respectively. Both N and C-SrtA have a C-terminal HIS tag, by virtue of which they were purified (see Figure 60 19). In addition the N-SrtA has a pelB sequence that allows it to be exported to the periplasm of the bacteria, once exported the export sequence is cleaved off. C-SrtA and N-SrtA expressed as well as the wild type SrtA (was cloned into pDEST17 by a colleague in the lab). SrtA and C-SrtA migrate just above the 24 kDa band of the protein ladder (see Figure 19) in a SDS-PAGE gel. However two very close bands were observed for the N-SrtA, both equal in intensity, one of them the periplasmic protein (with the pelB sequence cleaved off) and the other the cytoplasmic full length protein. The pelB sequence is about 22 aa long, ~ 2.2 kDa and is clipped off by the residential proteases once transported into the periplasm. The proteins N/C-SrtA were purified in good yields using the Ni-NTA-column. To determine if N- and C-terminal extensions perturb the function of SrtA, N/C-SrtA were incubated with EGFP-LPETG and GG-TMR , native SrtA catalyses the transfer of Gn , n = 1 to 5 , to the LPETG motifs of proteins by cleaving in between the T-G of LPETG and ligating the Gn peptide to threonine. The activity assay was carried out with appropriate positive and negative controls (see Figure 20), and it was found that the activity of N/C-SrtA was comparable to the wildtype the active site mutants were not active as expected, in that they were unable to transfer GG-TMR onto EGFP-LPETG even after 12 hrs of incubation at 37 °C. Also the hydrolysis and transpeptidation activities of C-SrtA were measured independently using quenched fluorescent probes (see Figures 21 and 22). As judged from the RFU, activity of C-SrtA was a bit lower than that of SrtA, but this could also be due to the minor differences in concentration of the two proteins used for the experiment. 61 3B.4 Self-ligation assay of N/C-SrtA In order to test the concept that SrtA with an N-terminal GGGSE extension can transfer a LPETG peptide onto its own N-terminal GGG- extension and that C-SrtA can catalyze the transfer of GG peptide onto its own C-terminus, the enzymes were incubated with their respective substrates bound on SA beads. In the case of N-SrtA it was LPETG-Biotin and in the case of C-SrtA it was G5-BIOTIN. After the reaction the beads were boiled in buffer R2 and run on a SDS PAGE gel. Upon probing with anti-biotin antibody it was evident that N/C-SrtA could catalyze the transfer of their substrates onto themselves, while the active site mutants lacked the activity (see Figure 23). As expected a single band was observed in the case of N-SrtA. As noted previously during the purification of N-SrtA two closely spaced bands were observed in the SDS-GEL. The bigger of them, SrtA with the pelB sequence does not contain an N-terminal glycine and the N- terminus would be too far away from the active site for any catalysis. This proved that the concept of capturing active enzymes using their substrates as tags could be extended to the phage system. 3B.5 Display of N/C-SrtA on phages N-SrtA/C-SrtA genes were cloned into vectors that will enable their expression on the phage. Cloning of N-SrtA followed regular cloning procedures and it was cloned in to pFAB5c.HIS phagemid, while C-SrtA had to be cloned into the T7 genome (see Figure 11) 10-3-B. Recombinants were identified using a PCR screen, sent in for DNA sequencing and verified. While the production of T7 phages is straightforward, 62 25kDa SrtA SrtA* C-SrtA C- N-SrtA N-SrtA* pelB +SrtA 25kDa Figure 19: Purification of SrtA and the different versions of SrtA. SrtA and the N- and C-terminal extensions of SrtA (C-SrtA and N-SrtA), purified via Ni-NTA column, run on a SDS page gel and coomasie stained. Note: twin bands get purified during NSrtA purification. 25kDa 1 2 3 4 5 Figure 20: Activity assay of SrtA, N-SrtA and C-SrtA as detected by in gel fluorescence scanning. 1: N-SrtA, EGFP-LPETG and GG-TMR. 2: N-SrtA-C184A, EGFP-LPETG and GG-TMR. 3: C-SrtA, EGFP–LPETG and GG-TMR. 4: C-SrtAC184A, EGFP–LPETG and GG-TMR. Fluorescence visualized by running the samples on a SDS-PAGE gel and scanning using the appropriate wavelength in a Typhoon scanner. 63 to package pFAB5c.HIS-N-SrtA into phages, the host cells carrying the phagemid is infected with helper phages. The phagemid is packaged into helper phage coat proteins preferentially over the helper phage genome. Large scale cultures of SrtAM13 phage and SrtA-T7 phage were produced and PEG precipitated. It was not possible to detect the expression of N-SrtA as the cloning scheme did not facilitate the addition of any affinity tag (it was required to keep the N-terminus free). But it was possible to introduce a C-terminal affinity tag into C-SrtA, accordingly a HIS tag was introduced. Expression of SrtA on the T7 phage was verified using a plaque lift method and regular western blotting procedures (see Figures 24 and 25). 3B.6 Activity assay of SrtA on phage It is well known that many enzymes when displayed onto the surface of phages do not retain their activity or the activity drops down to some extent [3]. For the selection scheme to work however it is imperative that the SrtA displayed on phage retains its activity to an extent that is detectable. N/C-SrtA on phage should be able to catalyze the same functions like N/C-SrtA in solution. PEG precipitated C-SrtA-T7, N-SrtAM13 phages were incubated with EGFP-LPETG-V5 and GG-TMR, and analyzed for fluorescence by separating the components in a SDS gel and scanning using the typhoon scanner. A faint but definite fluorescence band corresponding to the size of the 10B-SrtA-LPETG-G-TMR fusion and EGFP-LPETG-G-TMR was observed (see Figures 25-27), which was absent in SrtAC184A, displayed on the T7 phage. No band corresponding to the EGFP-LPETG-G-TMR was observed when EGFP-LPETG and GG-TMR were incubated with N-SrtA-M13-phage. 64 RFU A1 A1: SrtA B1: C-SrtA C1: C-SrtA-C184A D1: no enzyme B1 C1 D1 Time in sec RFU Figure 21: Transpeptidation of SrtA and C-SrtA as measured using quenched fluorescent substrates. SrtA (A1), C-SrtA (B1), C-SrtA-C184A (C1), or no enzyme (D1) was incubated with quenched fluorescent substrate (ACC-LPETG-DABCYL) and GG-peptide to measure the transpeptidation activity of SrtA. When kinetics was followed through the ACC channel in a microplate fluorescence reader, definitive increase in fluorescence was observed in the case of A1 and B1, whereas fluorescence of C1 and D1 remained in the background levels. A1: SrtA B1: C-SrtA C1: C-SrtA-C184A D1: no enzyme A1 B1 C1 D1 Time in Sec Figure 22: Hydrolysis activity of SrtA and C-SrtA as measured using quenched fluorescent substrates. SrtA (A1), C-SrtA (B1), C-SrtA-C184A (C1), or no enzyme (D1) was incubated with quenched fluorescent substrate (ACC-LPETG-DABCYL) to measure the hydrolysis activity of SrtA. When kinetics was followed through the ACC channel in a microplate fluorescence reader, definitive increase in fluorescence was observed in the case of A1 and B1, whereas fluorescence of C1 and D1 remained in the background levels. 65 1 2 3 4 73 kDa 25 kDa Figure 23: Self ligation assay of N-SrtA and C-SrtA as detected by anti-biotin western blot. 1: C-SrtA with G5-biotin on SA-beads. 2: C-SrtA-C184A with G5-biotin on SAbeads. 3: N-SrtA with Biotin-LPETG on SA-beads. 4: N-SrtA-C184A with BiotinLPETG on SA-beads. Biotin was detected using anti-biotin western blot. Red arrow: A higher molecular weight band was always seen with C-SrtA both during purification and activity assay, which was absent from C-SrtA-C184A. Molecular weight indicates that it could be a dimer. (For concentrations of each component in the assays, please refer to materials and methods) Figure 24: Plaque lift: C-SrtA-T7-Phages were lifted onto nitrocellulose membrane and probed with anti-HIS antibody. 73 kDa 1 2 3 4 5 Figure 25: Expression levels and activity assay of C-SrtA on the T7 phage as detected by anti-biotin western blot. 1: C-SrtA-10B-fusion, 2: C-SrtA-C184A-10B-fusion, 3: C-SrtA-C184A-10B-fusion incubated with GG-TMR, 4: bare T7 phages incubated with GG-TMR, 5: C-SrtA-10B-fusion incubated with GG-TMR. Red arrow: upon ligation with GG-TMR, the 6X-HIS tag is lost from the C-SrtA-10B-fusion. 66 3B.7 Enrichment of SrtA-phages from a pool of bare phages While whether or not N-SrtA on M13 was active remained a question, it was decided to do further experiments with the C-SrtA. The T7 SrtA phages were diluted into bare T7 phages (T7 wildtype phages). The HIS tag on SrtA, as well as activity of SrtA was used to enrich the SrtA phages from the wildtype T7 phages. 3B.7.1 Affinity based enrichment The SrtA phages on T7 were diluted at 1 in 104 or 1 in 105, into non-SrtA wildtype phages. This mixture was applied to the Ni-NTA beads (washed and blocked with BSA), and incubated at 4 °C for 1 hr. After removal of the unbound phages, the phages were subjected to amplification. This was continued until 6 rounds. After every round a PCR (using primers that amplified out the SrtA gene specifically) was done using the amplified pool of phages as template. After 3 rounds, enrichment was evident and became complete in six rounds (see Figure 28). 3B.7.2 Activity based enrichment The SrtA phages on T7 phages were diluted at 1 in 102 or 103, in non-SrtA phages. This mixture was applied to the G5-Biotin bound SA beads, and incubated at 37 °C for 2 hrs. After removal of the unbound phages, the phages were subjected to amplification. This was continued until 6 rounds. After every round a PCR using primers that amplified out only the SrtA gene was done using the amplified pool of phages as template. Unlike the affinity based enrichment, activity based enrichment how ever did not occur even at the end of 6 rounds, extending the number of rounds to 13 had no effect either. Other experimental conditions were tried but without any 67 effect (see Figure 29). As can be seen from Figure 29b, no specific enrichment was detected in any of the conditions tried. When non stringent wash conditions were used, a faint band corresponding to the size of the SrtA gene was found in all samples (see Figure 29b), despite the absence of the G5-Biotin peptide in the negative control samples. The reason why we failed to achieve activity based enrichment may be that, the activity of SrtA on phage was too low to begin with to allow any significant binding during the 1st round of enrichment. Moreover high background levels of binding of phage to SA beads might have made it impossible to detect any low amounts of enrichment that may have taken place (see Figure 29a). 68 75kDa 10B-SrtA-LPETGG-TMR 25kDa Figure 26: Activity assay of C-SrtA on T7 phage: 1: C-SrtA-T7phage, GG-TMR and EGFP-LEPTG-6XHIS. Red arrow indicates EGFP-LPETG-GG-TMR (For concentrations of each component in the assays, please refer to materials and methods). 1 2 3 4 5 6 7 Figure 27: Self ligation assay of C-SrtA on T7 phage: 1: T7phage, GG-TMR, 2: CSrtA-C184A-T7phage, GG-TMR, 3 to 7: decreasing concentrations of GG-TMR incubated with C-SrtA-T7phage. M 1 2 3 4 5 6 1 2 3 4 5 6 +ve -ve Figure 28: Affinity enrichment of C-SrtA-T7-phage. M: 1 kB DNA ladder, NEB. 1 to 6: rounds 1 to 6 of affinity enrichment (1 in 104, i.e., 1 C-SrtA-T7-phage in 104 bare T7 phages). 1 to 6: rounds 1 to 6 of affinity enrichment (1 in 105, i.e., 1 C-SrtA-T7phage in 105 bare T7 phages). Enrichment detected using PCR (primers T7up and T7down). 69 M 1 2 3 4 5 6 7 8 a b Figure 29a: Activity based enrichment of C-SrtA-T7. Odd lanes, C-SrtA-T7:T7, 1:100, with G5-Biotin. Even lanes: C-SrtA-T7:T7, 1:100, without G5-Biotin. Lanes 1 to 8 represent 4 rounds of enrichment. Lane a: C-SrtA-T7:T7, 1:100, with G5-Biotin in round 0 of enrichment. Lane b: C-SrtA-T7:T7, 1:100, without G5-Biotin in round 0 of enrichment. Lane M: 1Kb ladder from NEB. Experiment was done using less stringent wash conditions. M 1 2 3 4 5 6 7 8 Figure 29b: Activity based enrichment of C-SrtA-T7. Odd lanes, C-SrtA-T7:T7, 1:100, with G5-biotin. Even lanes: C-SrtA-T7:T7, 1:100, without G5-Biotin. Lanes 1 to 8 represent 4 rounds of enrichment. Input material to the experiment was the same as used in Figure 29a. Lane M: 1Kb ladder from NEB. Experiment was done using stringent wash conditions. 70 3C Detection of binders of 3CL protease from a phage library The 3CL protease was an important drug target for the Severe Acute respiratory Syndrome which had its origins in China and spread to other parts of the world. Several groups have been engaged in the discovery of inhibitors for the viral protease yet no significant progress has been made. In this project, determining inhibitors of the protease using the phage display approach was considered. While the traditional method of identifying peptide inhibitors was through the construction of a large peptide libraries and scanning for inhibition using a suitable assay, as a quicker alternative it was decided to scan a commercially available random peptide phage display library for good binders to the active site mutant of the viral protease. Incubating the library with the active enzyme will cleave most of the binders, but incubation with the active site mutant will enable isolation of binders. Upon emergence of a strong binder a group of similar peptides maybe then be designed, synthesized and the inhibition of the protease can be studied in solution. 3C.1 Biopanning of a model protein Streptavidin A random 7 mer peptide library on the surface of the filamentous phage was purchased from NEB. The library contains peptides displayed as N-terminal fusions to the capsid protein gene III of the M13 phage. Also the M13 phage coded for the enzyme β-Galactosidase and hence the library phages appear blue upon X-Gal selection. Because of the troublesome contamination of the library phages with environmental M13 phages, this blue/transparent selection served useful to identify library phages from contaminating environmental phages at all times. In order to optimize the biopanning assay conditions, at first a biopanning experiment with the protein Streptavidin was undertaken. In searching for peptide binders to SA from a 71 random peptide library on the microarray, some groups have reported a strong binding preference to the peptide sequence “HPQ” [111]. The phage library contained 2 x 109 electroporated clones, amplified once to give a total of 70 copies of each sequence as opposed to the total possible 20 x 107 =1.28 x 109 sequences. 96-well SA high binding capacity plates from PIERCE were found to be convenient for all the procedures of biopanning. As a negative control to the experiment, the library was also panned against BSA coated in polystyrene wells. Every round was characterized by 3 steps, incubation of a definitive number of phages (1 x 1011 phages which represent 100 copies of every sequence in the library) with the SA plates; washing to remove the unbound phages; elution of bound phages done using glycine-HCL, pH 2.2, and neutralization of the pH with Tris-buffer (see Figure 3). After the elution the phages were amplified for the next round of selection. After every round of selection the input and the output titers were counted. As compared to the negative control the titers from the SA plates were a 100 fold higher. This was consistently observed in all the three rounds, while the titers from the BSA plates were roughly the same in all the three rounds. After three rounds individual plaques were picked and surveyed for binding to SA using the binding assay. 3C.2 Binding assay to detect the strongest of binders After three rounds of selection individual plaques were picked and inoculated to amplify them separately. 24 individual clones thus obtained were incubated in SA microtiter plates, 1 x 109 of each per well. After incubation for an hour at RT, the wells were washed to remove unbound phages and the bound phages were eluted as described in Section 3C.1 and titered. The percentage of input phages recovered was calculated. Amongst the different clones 16 representative clones were sequenced. 72 Clone No Input % Input No of residues recovered conserved Output Sa_1 6 x 1010 1.4 x 107 0.02 3 Sa_2 4 x 1010 9.1 x 107 0.214 1 Sa_3 4 x 1010 ND -- 7 Sa_4 1 x 1010 7 x 106 0.006 3 Sa_5 ND 2.5 x 106 -- 1 Sa_6 4 x 1010 1.75 x 106 0.001 3 Sa_7 ND 1 x 107 -- 0 Sa_8 7 x 1010 5.25 x 106 0.007 2 Sa_9 2 x 1010 2.88 x 106 0.01 0 Sa_10 2 x 1011 1.66 x 106 0.0008 2 Sa_11 ND 1.05 x 106 -- 3 Sa_12 ND 1.4 x 108 -- 0 Sa_13 4 x 1010 5.25 x 105 0.0001 0 Sa_14 1 x 1010 1.4 x 108 1.4 7 Sa_15 1 x 1010 7 x 105 0.007 2 Sa_16 0.5 x 1010 1.2 x 108 2.4 7 Table 1: Results from the binding assay from biopanning against SA 73 They contained both strong binders (> 0.1% input pfu recovered) and weaker binders (> 0.1% input pfu recovered) (see Table 1). The sequencing results revealed the presence of the strongly conserved motif “HPQ“ in some of them (see Table 3). In an independent experiment which was done using far more stringent washes (0.5% Tween 20), most of the 8 clones sequenced were found to contain the “HPQ” consensus (see Table 2). Thus the power of the phage display approach was demonstrated in that, it was possible to fetch out a single peptide sequence from a billion. Also the protocols for biopanning were thus optimized. 3C.3 Expression and Mutation of the 3CL protease The clone bearing the 3CL protease, pGEX-4T1-3CL was kindly given to us by Prof. Song Jian Xing of DBS, NUS. The vector allows fusion of cloned genes to GST protein, thus facilitating affinity purification using GSH columns. Using the method of full length plasmid amplification from STRATAGENE, to produce site directed mutations, the active site Cysteine 145 of the protease was mutated to Alanine (see Figure 12). The clone, pGEX-4T1-3CL-C145A, thus obtained was sequence verified and transformed into BL-21-DE3 cells. Upon induction of expression with IPTG, the protein was purified using GSH beads, run on the SDS-PAGE and was found to be > 95% pure (see Figure 30). 3C.4 Bio-panning against the 3CL protease mutant Having stabilized the conditions for biopanning, 3CL mutant was immobilized onto GSH beads (by means of its GST tag). Using the same library and similar panning conditions as in Section 3C.1, 3 rounds of the selection were carried out. The titers were obtained at the end of each round was compared with the negative controls (the 74 1 2 3 54kDa Figure 30: Expression levels of 3CL protease and 3CL protease mutant. 1: Crude cell lysate from un-induced lane, 2: Crude cell lysate of over expressed 3CL protease. 3: Crude cell lysate of over expressed 3CL-C145A. Clone number Sequence 1 S L I A H PQ 2 T L L A H PQ 3 H FW D H PQ 4 S L I N H PQ 5 H FW D H PQ 6 T L L A H PQ 7 L L W P S L P 8 ST G S T FW Table 2: Sequencing results of the peptides from biopanning against Streptavidin. 6 clones out of the 8 that were sequenced contained the strongly conserved motif HPQ, the rest showed conserved residues in various positions as indicated above in red fonts. This experiment was done using stringent wash conditions. 75 Clone No Sequence Sa_1 S Sa_2 W S W P R A M L F E W S Sa_3 N L V N H P Q Sa_4 N L Q F M P Y Sa_5 H P G N S Y N Sa_6 T E L Q S P D Sa_7 L G C A C C S Sa_8 T T I S P H V Sa_9 D F S W V S H Sa_10 S T W A Sa_11 S P V A P W P Sa_12 H T S P T F Sa_13 Sequencing results not good Sa_14 N L I N H P Q Sa_15 X L P H F Q Sa_16 S I A H P Q L L T A Y S T Table 3: Sequencing results of the peptides from the binding assay, biopanning against SA. 3 clones out of the 16 that were sequenced contained the strongly conserved motif HPQ, the rest showed conserved residues in various positions as indicated above in red fonts. This experiment was done using less stringent wash conditions. 76 phage library applied to the GSH beads). However no clear increase in titers as seen for the SA control experiment was visible. If the binders to the 3CL mutant were weak in binding affinity then no clear difference in titers between that of the protease and BSA bound beads could be expected. Hence more rounds of selection were carried out. At the end of 6 rounds, 24 clones were selected for binding assay against the 3CL mutant immobilized on ELISA plates. However upon counting the titers, many of the clones were found to have negligible binding, almost equivalent to that of the negative control (BSA immobilized on the ELISA plates). Nevertheless some of the binders were sequenced. None of the sequences obtained were similar to the substrates already known (see Table 4), and in corroboration to the binding assay results, no consensus sequence had been reached. The reason why we failed to isolate good and specific binders could be manifold. It was expected that atleast “substrate like” sequences would be isolated. Studies have established that the 3CL protease prefers a hydrophobic residue in the P2 position, a glutamine in the P1 position, and alanine, serine or glycine residue in the P1’ residue [106]. The probability of finding the reported substrate sequence “AVLQSGF” was calculated as per the suggestions given in the manual of the peptide phage display library. The formula suggests the existence of at least 3 such sequences in the library. During the course of the experiments, the library material obtained from the manufacturer was amplified once to give more material. However, once the library is replicated to produce more material the chances of finding all the sequences from the initial library are reduced. It largely depends on whether all the phages in the original library infected and replicated in the host cells. Also it might be possible that the library that was used contained no good binders at all. Many groups in fact custom-make dedicated peptide 77 libraries that includes the known binding preferences of the protease they study [6975]. Clone number Sequence 1 H Q P S R Q Y 2 L A H S RD P 3 I W A F A T P 4 S P P P P S M 5 A R V T E M S 6 Q H M T Q V T 7 Y P Y Q W P K 8 L P T L D R G 9 E S A P T S T Table 4: Sequencing results of the peptides from bio-panning against 3CLmutant. 78 4. Conclusions In this thesis three different approaches to protein engineering and modulation were evaluated. As one of the approaches to protein engineering, a strategy for the immobilization of proteins site-specifically via the N-terminus onto the microarray was developed. The chosen model proteins were cloned into a vector system that facilitates the expression of the protein with an N-terminal intein fusion. An extra cysteine residue was introduced at the junction of the intein and protein fusion. Upon expression of the intein-protein fusion, intein splices out, leaving the protein with an N-terminal cysteine. The proteins thus produced were applied onto thioesterfunctionalized slides for uniform orientation. As a complementary method, intein based approach to site-specifically biotinylate proteins derived from yeast on their Cterminus was successfully set up. Three different commercially available inteins were tested out. Amongst them one, the Sce VMA intein was chosen as the intein of choice because of its high yields and minimal in vivo cleavage. It was shown successfully that EGFP fused to Sce VMA intein and expressed from small scale cultures in yeast could be biotinylated at the C-terminus using the intein mediated thioester generation process. Further experiments were done to immobilize the biotinylated proteins onto avidin derived surfaces. Despite optimizing procedures the yields of biotinylated proteins was not sufficient to achieve immobilization on the microarray. While this problem could be solved by expressing the proteins in large scale cultures from yeast, it was not pursued further, as the protocol was not adaptable to high throughput immobilization. Hence it was concluded that obtaining biotinylated proteins in a high throughput manner from yeast, (e.g. in 96 well plates), was not feasible using this approach. 79 A selection scheme for evolving SrtA on phage has been setup. The selection scheme is a varied version of the product capture approach used to evolve enzymes on the phages. The selection scheme was at first proved by expressing SrtA with its Cterminus fused to its LPETG substrate (C-SrtA). When C-SrtA was incubated with G5-Biotin, it was able to catalyze the transfer of G5-Biotin onto its own C- terminus, thus proving the self-ligation ability of SrtA. C-SrtA was then successfully displayed on the phage and its activity was proven. Finally C-SrtA-T7 proved capable of ligating GG-TMR onto itself, thus demonstrating the success of the selection scheme. However the activity of SrtA on phage was such that we were unable to enrich it based on its activity from a pool of bare phages. Nevertheless, using this selection scheme and a library of random mutants of SrtA, enzymes with improved activity may be selected for in the future. A suitable screening method may then be used to identify the most active members. The screened pool can also be subjected to further rounds of mutagenesis and the selection might be repeated. The whole process can be continued until SrtA with desired activity evolves. A peptide library on phage was scanned for inhibitors to the SARS viral protease 3CL. Using the affinity selection method that is routinely used to biopan peptide libraries on phage, a commercially available peptide library was scanned for affinity binders to the mutant 3CL protease immobilized on GST beads. 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Buffer H4 50 mM NaH2PO4·H2O 300 mM NaCl 250 mM Imidazole pH to 8.0 using NaOH. Buffer H5 50 mM Tris base 150 mM NaCl pH to 7.5 using HCL. 96 Buffer G1 20 mM NaH2PO4·H2O 100 mM NaCl 0.1 % v/v Tween-20 1 mM EDTA 1 mM DTT pH 7.5 Bufffer G2 137 mM NaCl 3 mM KCl 8 mM NaH2PO4 1.5 mM KH2PO4 pH 7.4 with HCL. Buffer G3 10 mM Tris 50 mM GSH BUFFER G4 10 mM Tris 1 mM DTT 1 mM EDTA pH 7.4 97 Buffer C1 20 mM Tris 500 mM NaCl 0.1 %v/v Tween -20 pH 7.5 Buffer C2 20 mM Tris 1M NaCl 0.1 % v/v Tween -20 pH 8 Buffer C3 30 mM MESNA 1 mM EDTA 20 mM Tris 500 mM NaCl pH 8-8.5 Buffer R1 50 mM Tris base 150 mM NaCl 5 mM CaCl2 2 mM 2-ME Adjust pH to 7.5 using HCl. 98 Buffer R2 10 % v/v Glycerol 50 mM Tris (pH 6.8) 2 mM EDTA (pH 8) 2% SDS 100 mM DTT Pinch BPB Buffer C 20 % v/v PEG 2.5 M NaCl Autoclave and store at RT Buffer D 50 % PEG Autoclave and store at RT Buffer E 10 mM Tris (pH 6.8) 1 mM EDTA (pH 8) 4M NaI Store at RT in the dark. 99 Buffer F 50 mM NaH2PO4·H2O 300 mM NaCl Adjust pH to 8.0 using NaOH. Buffer G 0.2 M Glycine 1 mg/mL BSA pH 2.2 with HCl. Buffer H 0.1 M NaHCO3 (pH 8.6) 5 mg/ml BSA Filter sterilize, store at 4°C. 2XYT broth (per liter) 20 g Tryptone 10 g Yeast Extract 5g NaCl Autoclave and store at RT 100 10X SD-URA (per liter) 10 g Yeast nitrogen base 29.311 g Ammonium Sulphate 0.12 g Adenine Sulphate 0.12 g L-Arginine (HCL) 0.12 g L-Histidine (HCL) 0.18 g L-Lysine(mono-HCL) 0.12 g L-Methionine 0.3 g L-phenylalanine 0.12 g L-trptophan 0.18 g L-tyrosine 0.9 g L-Valine 0.36 g L-Leucine 0.18 g L-Isoleucine The above components were dissolved in de-ionized water and filter sterilized Just before use, the 10X media is diluted to 1X in sterile de-ionized water and 2 % glucose is added to it. 101 APPENDIX B Cloning table: List of constructs, primers and primer features. NAME OF THE CLONEA SEQUENCE OF PRIMERS FEATURESC NAME OF THE PRIMERS pDONOR201-SrtA,B 5’GGG GAC AAG TTT GAA AAA AGC AGG CTT Staphylococcus aureus genomic TAC CAT GCA AGC TAA ACC TCA A 3’ DNA, 5’GGG GAC CAC TTT GTA CAA GAA AGC TG GGT (pDEST-17-SrtAB) TTC ATT TGA CTT CTG TAG C 3’ pDONOR201-EGFP-LPETG-V5,B 5’GGG GAC AAG TTT GTA CAA AAA AGC AGG AttB1 SrtA-Attb1 AttB2 SrtA-Attb2 AttB1 EGFP-V5-Attb1 EGFP-LPETG pEGFP, CTC AAT GGT GAG CAA GGG CGA G 3’ (pDEST-17-EGFP-LPETG-V5B) 5’AGG GAT AGG CTT ACC AAG GCC GGT TTC CGG LPETG sorting AAG GGA 3’ motif 102 5’GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT AttB2 , V5 V5-Attb2 TCA CGT AG AAT CGA GAC CGA GGA GAG GGT TAG GGA TAG GCT TAC C 3’ pFAB5c.HIS-N-SrtA, 5’ATT AGG CCC AGC CGG CC GGT GGC GGA TCT Sfi I site, N Staphylococcus aureus genomic GAA GGC GGA GGA AGT GAG GGA GGT GGA AGC terminus extension. DNA. GAA CAA GCT AAA CCT CAA ATT CCG 3’ (pFAB5c.HIS-N-SrtA-EXPF) 5’ GCC GTA ATT AGC GGC CGC TGC TTT GAC SfiI-Linker-15aaSrta- Fwd Not I site. Not1-SrtA-Reverse SrtA-C184A-Fwd TTC TGT AGC TAC AAA GAT TTT ACG TTT TTC CCA AAC GCC TG 3’ pFAB5c.HIS-N-SrtA-C184AG; 5’ CAA TTA ACA TTA ATT ACT GCT GAT TAC AAT Bases that were pFAB5c.HIS-N-SrtA-C184A- GAA AAG 3’ mutated EXP; 103 pDEST17-C-SrtA-C184A; 5’ CTT TTC ATT GTA ATC ATC AGC AGT AAT TAA Bases that were pTOPO-C-SrtA-C184A, (10-3-B- TGT TAA TTG 3’ SrtA-C184A-Rev mutated C-SrtA-C-184A). pTOPO-C-SrtA, Staphylococcus 5’ GGC GGT GGC GGT GGC CAA GCT AAA CCT Linker sequence Linker SrtA fwd aureus genomic DNA. CAA ATT CCG AAA 3’ (1)D (10-3-B-C-SrtA). 5’ GCATTATAT GGA TCC CGGC GGT GGC GGT BamH1,Linker BamH1-Linker – GGC CAA GCT AAA 3’ (2)D sequence Fwd 5’ GCAA CCCGGG TTA GTG ATG GTG ATG ACC Xma1 site, HIS tag LPETG-6HIS- GGT TTC CGG CAG 3’ (3)D LPETG STOP-Xma1-Rev 5’ ACC GGT TTC CGG CAG ACC ACC TTT GAC LPETG, linker. LPETG-GG-SrtA- TTC TGT AGC TAC A 3’ (4)D Rev 104 pDONOR-C-SrtA, Staphylococcus 5’ GGG GAC AAG TTT G TA CAA AAA AGC AGG Attb1 sequence ATTB1 -SRTA - aureus genomic DNA. CTT TAC CAT G CA AGC TAA ACC TCA A 3’ (1)E (pDEST17-C-SrtA) 5’ ACC GGT TTC CGG CAG ACC ACC TTT GAC LPETG sorting TTC TGT AGC TAC A 3’ (2)E motif, linker LPETG-GG-SrtA- 5’GGGG AC CAC TTT GTA CAA GAA AGC TGG GTC Attb2 sequence Rev Fwd TTA ACC GGT TTC CGG CAG 3’ (3)E pYESDEST-52-Egfp-Intein-1, 5’GGG GAC AAG TTT GTA CAA AAA AGC AGG pTwin1-EGFP CTT CGA AGG AGA TAG AAC CAT GGT GAG LPETG - attb2 Attb1 sequence attB1-EGFP-inteinF CAA GGG CGA GGA G 3’ 5’GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC Attb2 sequence AttB2-EGFP- TCA TTG AAG CTG CCA CAA GGC 3’ intein-R 105 pYESDEST-52-Egfp-Intein-2, 5’GGG GAC AAG TTT GTA CAA AAA AGC AGG pTWIN2-EGFP CTT CGA AGG AGA TAG AAC CAT GGT GAG Attb1 sequence AttB1-EGFPintein-F CAA GGG CGA GGA G 3’ 5’GGG GAC CAC TTT GTA CAA GAA AGC TGG Attb2 sequence GTC TCA TTG AAG CTG CCA CAA GGC 3’ AttB2-EGFPintein-R pGEX-4T-1-3CL-MUT, pGEX- 5’ CAT TAA AGG TTC TTT CCT TAA TGG ATC AGC Bases that were 4T-1-3CLI. AGG TAG TGT TGG TTT TAA CAT TGA TTA TG 3’ mutated 5’ CAT AAT CAA TGT TAA AAC CAA CAC TAC CTG Bases that were CTG ATC CAT TAA GGA AAG AAC CTT TAA TG 3’ mutated 3CL-C145A-FwdI 3CL-C145A-RevI 106 pFAB5c.HIS 10-3-B SrtA 5’ GCT TCC GGC TCG TAT GTT GTG 3’ Forward pFAB5c.HIS- Sequencing primer Fwd 5’ GGA GCT GTC GTA TTC CAG TC3’ Forward primer T7 Up 5’ TAA ACG GGT CTT GAG GGG TT 3’ Reverse primer T7 Down 5’ GTA AGT ATA AAA TGA CAA GTA 3’ Mid sequencing pFAB5c.HIS-srtA primer A. Name of the clone includes vector name and the gene cloned into the vector and source of the gene is in italics and is underlined. The name of the subclones, derived from the parental clones is given in red. Subcloning was done either using RE based methods or using gateway methods. B. Constructs were made by colleagues in the lab, included in thesis because I made use of it. C. Features of the primers are highlighted as follows, Affinity tags in blue, Restriction enzyme sites in bold, linker sequences underlined. D. PCR was done at first using primers 1 and 4, the resultant PCR product was used for PCR using primers 2 and 3. E. PCR was done at first using primers 1 and 2, the resultant PCR product was used for PCR using 1 and 3. F. The Digestion of the pFAB5c.HIS-N-SrtA with EagI gets rid of the Gene III from the vector and converts it into a expression vector, pFAB5c.HIS-N-SrtA-EXP. The vector is now ready for expression of cloned fragment. Gene III is toxic to the bacterial cells if over expressed by IPTG induction and hence it is required to be removed before over expression from the lac promoter. G. All constructs given in this section were made using full plasmid PCR amplification using the same set of primers, to mutate the active site of SrtA. H. For details on PCR, SDM, TA cloning, gateway cloning and RE based cloning see Chapter 2 of thesis. I. Construct and primers gifted by Prof. Song Jian Xing. 107 Expression table: Expression conditions of different expression constructs. NAME OF THE EXPRESSION CLONE HOST pDEST-17-SrtA BL-21-AI EXPRESSION CONDITIONS OD600 = 0.5, 0.2 % arabinose, 37 °C, 4 hrs WESTERN BLOT CONDITIONS Penta-HIS-HRP conjugate, 1/1000 dil in milk, 1hr, RT, 10 min each, 6 times, PBST (Tween 20, 0.1%) pDEST-17-EGFP- BL-21-AI OD600 = 0.5, 0.2 % arabinose, RT, O/N V5 Penta-HIS-HRP conjugate, 1/1000 dil in milk, 1hr, RT, 10 min each, 6 times, PBST (Tween 20, 0.1%) pFAB5c.HIS-N-SrtA XL-1-BLUE, pFAB5c.HIS-N- ER2738 OD600 of 0.5, 1 mM, IPTG, 37°C , 4 hrs Penta-HIS-HRP conjugate, 1/1000 dil in milk, 1hr, RT, 10 min each, 6 times, PBST (Tween 20, 0.1%) SrtA-C184A pDEST17-C-SrtA pDEST17-C-SrtA- BL-21-AI OD600 = 0.5, 0.2 % arabinose, 37°C, 4 hrs Penta-HIS-HRP conjugate, 1/1000 dil in milk, 1hr, RT, 10 min each, 6 times, PBST (Tween 20, 0.1%) C184A 108 pYESDEST-52-Egfp- Yeast- InvSC- Grown in SD-URA + glucose media, OD600 = 1°antibody: anti-CBD, 1/5000 dil in milk, 2° Intein-2 antibody: anti-Rabbit -HRP in milk, 1/ 5000 dil in 1 (Invitrogen) 0.4, 2 % galactose, SD-URA, 30 °C, 24 hrs milk, 1 hr RT , 10 min each, 6 times, PBST (Tween 20, 0.1%) pYESDEST-52-Egfp- Yeast -InvSC- Grown in SD-URA + 2 % glucose media, 1° antibody: anti-CBD, 1/5000 dil in milk, 2° Intein-1 OD600 = 0.4, 2 % galactose , SD-URA , 30°C, antibody: anti-Rabbit-HRP,1/ 5000 dilution in 24 hrs milk, 1 hr RT, 10 min each, 6 times, PBST 1 (Invitrogen) (Tween 20, 0.1%) pGEX-4T-1-3CL BL21-DE3 OD600 of 0.5, 0.5 mM IPTG, RT, O/N Anti-GST HRP conjugated, 1/5000 dil in milk, 1 pGEX-4T-1-3CL- hr, RT, 10 min each, 6 times, PBST (Tween 20, Mut 0.1%) 109 [...]... different approaches to protein engineering and modulation that were evaluated in this report In this report, three different approaches to protein engineering and modulation were evaluated As one of the approaches to protein engineering, a strategy for the immobilization of proteins site-specifically via the N-terminus onto the microarray was developed The chosen model proteins were cloned into a vector... Protein engineering and modulation are used in the following context throughout the thesis and are defined as, “Processes of modifying the structure of proteins or introducing unnatural functionalities to create tailor-made proteins serving useful applications” Several methods that exist to modify and engineer proteins can be broadly grouped into 2 different categories (a) Rational design and Protein. .. Protein engineering Proteins are the most important work horses in the cells; they serve myriad functions and are also important structural determinants within cells Ability to modulate and engineer proteins serves as important tools to understand their structure and function [1], it can also give rise to useful proteins that can fulfill biotechnological and industrial applications [2] The terms Protein. .. impaired origin of replications) When using the phagemid method to display proteins, depending on the size of the protein and how well it is tolerated on the phage, the number of copies of the displayed protein can vary from 0-5 per phage 9 Intein fusion construct EGFP INTEIN CBD Transformation into yeast cells MESNA mediated cleavage and tagging of proteins with CysBiotin v A idin Capture onto chitin column... 55 17 Purification of EGFP-Intein 3 fusion from yeast small scale cultures 55 18 Effect of different concentrations of cys-Biotin on the biotinylation efficiency of EGFP purified from different hosts 55 19 Purification of SrtA and different versions of SrtA 63 20 Activity assay of SrtA, N-SrtA and C-SrtA as detected by in-gel fluorescence scanning 63 xi 21 Transpeptidation of SrtA and C-SrtA as measured... what structural changes will give rise to the desired 1 function These limitations can be overcome by taking the proteins through the process of protein evolution [4], which mimics the natural process of evolution in the laboratory test tube The key points of the protein evolution methods are mutagenesis and selection of the fittest A repertoire of random mutants of a desired gene is created using genetic... protein/ peptide of interest tethered to it The genetic information of the protein/ peptide resides inside the phage and is retrievable any time by a simple sequencing step As such the phage then is a coded, amplifiable and infinitely storable bead In the affinity selection method, the protein of interest is coated onto a solid surface and the phages bearing the random peptide libraries are applied to. .. sequence emerges (a group of binders with similar sequences) To cite a few interesting examples, using the method of affinity selection, a number of cloned SH3 domains were used to select ligands from a random peptide library Upon identification of the ligands, these were used to probe conventional cDNA libraries for protein that bind to the identified ligands In this manner 18 homologs of the SH3 domain were... C-SrtA were displayed on phage and the activity was tested Display on the M13 phage allows the N-terminus of the displayed protein to be free Display on the T7 phage allows the C-terminus of the displayed protein to be free For display onto the M13 phage, SrtA was fused to the N-terminus of gene III To display proteins on the T7 phage, SrtA was fused to the C-terminus of the capsid gene10B While we... field of bioimaging, where by specifically adding on fluorescent tags onto proteins, one can study protein dynamics, localization, cell movement and cell growth [20, 21] Site-specific modification of proteins has also found use in the field of microarrays, where adding on tags like biotin to a protein allows it to be specifically immobilized onto an avidincoated surface [10, 11, 22] 2 1.1.3 The three different ... 1.1.3 The three different approaches to protein engineering and modulation that were evaluated in this report In this report, three different approaches to protein engineering and modulation were... as biotin to a protein allow it to be specifically immobilized onto an avidin-coated surface Different approaches to protein engineering and modulation using the phage display method and the intein... directed mutagenesis of genes 28 2.8 Expression of different fusion proteins from different vectors and hosts 30 2.9 Western blot of proteins 32 2.10 Affinity chromatography of proteins 33 2.10.1