Microfluidics and microarray based approaches to biological analysis 4

Chapter CHAPTER INTEIN-MEDIATED BIOTINYLATION OF PROTEINS AND ITS APPLICATION IN A PROTEIN MICROARRAY 4.1 Introduction 4.1.1 Developing Microarrays of Functionally Active Proteins DNA microarray is currently the method of choice for high-throughput analysis of nucleic acids at their transcriptional level. However, it has been shown that the mRNA expression level in a cell does not correlate well with the abundance of proteins.1 To gain more insights into protein functions, a number of techniques2 and binding chemistry have been developed to immobilize small molecules,3,4 peptides,5,6,7 and proteins 8,9,10 in a microarray for high-throughput protein studies.11 As mentioned in chapter 3, various strategies have been developed for the site-specific immobilization of kinase substrates onto functionalized slides. However, proteins are more difficult to handle than peptides. Indeed, they are delicate, denature in ‘harsh environments’ and protein arrays are stable and useful only for a very short period of time. Consequently, different approaches have been developed to ensure they retain their activity. Schreiber et al dissolved proteins in a 60 % solution in order to keep them hydrated;8 whereas Zhu et al attached them to the surface of PDMS micro wells.9 However, in most cases, protein immobilization was achieved via their nucleophilic residues, resulting in random orientations of proteins on the glass surface, whereas an oriented immobilization on the slide surface is desired for optimal protein activity. Indeed, in order for arrayed proteins to retain their full biological activity, they need to be arrayed in a proper orientation to ensure accessibility of their active sites with interacting molecules. 98 Chapter Thus far, there has only been one report of site-specific attachment of proteins on glass slides.12 Approximately 6000 yeast proteins were expressed as His-tag fusions, spotted onto Ni-NTA functionalized slides, and >80 % were found to retain their full biological activities, presumably as a result of site-specific immobilization which ensures most proteins on the slide to be oriented correctly. However, the binding between Ni-NTA and His-tag proteins is neither very strong, nor very stable and susceptible to interference by many commonly used chemicals,13 making this immobilization method incompatible with many protein screening assays. Besides the site-specific immobilization, another problem when working with protein microarrays is the high throughput expression and purification of proteins. So far, all protein arrays involve commercially available proteins or recombinant proteins.9 In the second case, proteins need to be purified before spotting, implying long, and laborintensive protocols. The in vitro expression of proteins appears as a promising solution and a protein array of in situ synthesized proteins has been reported.14 He at al developed a new protein array production procedure termed PISA (protein in situ array). PISA is designed to generate protein arrays directly from PCR generated DNA via cell-free protein synthesis and simultaneous in situ immobilization of the generated proteins on a surface. However, this array consists of microwells rather than a real array and this strategy has not been applied to the array format yet. In addition, post translation modifications may be lacking for in vitro synthesized proteins, and new strategies are required for the in vivo synthesis, purification and functionalization of proteins for site-specific immobilization. 99 Chapter 4.1.2 Site-Specific Protein Biotinylation On the contrary to the interaction between His tag and Ni-NTA previously used for protein microarrays, the biotin-avidin interaction is one of the strongest known noncovalent interaction.15 It is very stable toward a variety of harsh conditions,16 and has been widely used in standard biochemical assays for immobilization purposes. The in vivo and in vitro biotinylation of proteins have previously been reported,17,18 but with limited success due to low yields and non-specific nature of the biotinylation reaction.19 In some cases biotinylation resulted in the addition of a long peptide to the target protein, which may interfere with proper folding of the protein.20 Furthermore, avidin is known to be toxic to cells, making the expression of avidin-fused proteins a difficult task.21 4.1.3 Intein-Mediated Protein Engineering Inteins are naturally occurring proteins that are involved in the precise cleavage and formation of peptide bonds in a process known as protein splicing. The mechanism of protein splicing (Figure 4.1) was elucidated using a combination of site directed mutagenesis and chemical analysis and allowed the rational engineering of inteins for use in protein chemistry. The process of protein splicing involves the excision of an intervening protein sequence (the intein) from a precursor protein with the concomitant fusion of the two flanking protein regions through a native peptide bond. Controlled cleavage at single intein splice junctions led to the development of fusion protein purification on chitin columns.22 In so-called “native chemical ligation”23 described in chapter 3, an N-terminal cysteine containing peptide is chemically ligated to a second peptide possessing a thioester group with the resultant formation of a native peptide bond at the ligation junction. 100 Chapter Peptide thioesters for use in native chemical ligation are generated solely through chemical synthesis. The intein-based purification protocol has found wide applications in protein engineering where the expressed protein ligation (EPL) strategy is utilized to incorporate non coded amino acid sequences into a protein sequence (Figure 4.2 a).24,25 This strategy has been extended to modifications of proteins at their C-termini with a number of chemical tags.26 Recently, Tolbert et al reported the use of TEV protease to generate N-terminal cysteines from affinity-tagged fusion proteins (Figure 4.2),27 but unfortunately, the simultaneous protease cleavage and thioester labeling of the affinitytagged fusion proteins was unsuccessful since the thioester labels are inhibitors of the TEV protease, possibly because the TEV protease is a cysteine protease with an active site cysteine that can be acylated by the thioesters. HS HS N-extein C-extein Intein HS S HN Intein O O H 2N O O S O O N H NH2 S N H NH2 O O O O HS S N H H2 N Figure 4.1. Principle of intein-mediated protein splicing. 101 Chapter HS HS O + Protein NH3 O O Intein N H + NH3 His6 ENLYFQ Protein N H O- OO O HS O O O O + Protein NH3 Intein S + Protein H3N O- O- NH3+ O a O b HS SR R R +NH3 O HS HS O O R + NH3 Protein R N H O Protein N H OO O Figure 4.2. Generation of N-terminal cysteine proteins using TEV protease for (a) expressed protein ligation, (b) Generation of N-terminal 4.2 Results and Discussion 4.2.1 Intein-Mediated Biotinylation of Proteins at their C-Terminal1 As described in chapter 3, the native chemical ligation developed by Kent et al allows for the site-specific reaction between the N-terminal cysteine and the thioester function of any other compound. EPL represents a novel semisynthetic approach that has greatly expanded the utility of native chemical ligation chemistry and allows for the site-specific incorporation of non-coded amino acids into the protein of interest. By expressing a protein of interest as fusion to an intein, which also contains a chitinbinding domain for purification on chitin column, one can both purify and label at its C-terminal by flushing the column with the cysteine containing labeling agent. The expression and biotinylation of proteins was performed by Lue Yee Peng Rina, and cysteine biotin was synthesized by Dr Zhu Qing 102 Chapter Therefore, by reacting cysteine biotin with intein fused protein, one can in a single step, purify and site-specifically biotinylate proteins at their C-terminal (Figure 4.3). O HN O H2 N H N N H SH N NH S O N O O HS S S c) Biotinylation H N H2 N O b) Purification S NH HN O Spontaneous rearrangement S Chitin column N O N H N HS O O S N H Intein tag H N O O N H S HN NH O a) In vivo expression DNA Target protein Intein ta g Figure 4.3. Intein-mediated site-specific biotinylation of proteins Three proteins of interest, namely MBP (Maltose Binding Protein), EGFP (Enhanced Green Fluorescent Protein) and GST (Glutathione S-Transferase) were chosen as models and expressed in vivo as fusion proteins with an intein tag (intein fused to chitin binding domain) at their C-termini. The proteins were purified and biotinylated, in a single step (Figure 4.3), by first loading the crude cell lysate onto a column packed with chitin beads, then flushing the column with biotinylated cysteine (Figure 4.4), to obtain the C-terminally biotinylated proteins. 103 Chapter HS H N O S HN N H NH2 O NH O Figure 4.4. Cysteine Biotin used for Intein-Mediated Site-Specific Biotinylation The site-specific biotinylation of the proteins was unambiguously confirmed by SDSPAGE (Figure 4.5, a) and western blotting (Figure 4.5, b). Based on SDS-PAGE, the biotinylation reaction took place with 90-95 % efficiency, generating proteins in sufficient purity (> 95 %). Labeling of proteins expressed as intein fusion is very simple since the ligation reaction can be combined with affinity purification, allowing C-terminally modified proteins to be obtained from crude bacterial lysates in a single step. (a) (b) 10 Figure 4.5. MBP purification and biotinylation. (a) SDS-PAGE. (1) protein marker, (2) uninduced cell extract, (3) induced cell extract, (4) flow-through from column loading, (5) flow-through from column wash, (6) proteins bound to chitin column before cleavage, (7) flow-through from quick flush of cleavage agent, (8-9) first two elution fractions after overnight incubation at °C with cysteine biotin (10) remaining proteins bound to chitin column after cleavage. (b) Western blotting of biotinylated MBP. Biotinylated MBP was run on SDS PAGE and after transfer was detected using Streptavidin-HRP. 104 Chapter 4.2.2 Microarrays of Site-Specific Immobilized Active Proteins After biotinylation, without any further purification, the three biotinylated proteins were spotted directly, without any further treatment, onto an avidin-functionalized slide to obtain the corresponding protein array. (Figure 4.6) N HS O N H H N O O N H Immobilization S HN NH O STA slide Figure 4.6. Site specific immobilization of functionally active proteins A protein array was generated with the biotinylated EGFP, MBP and GST, and probed with Cy3-anti-EGFP, Cy5-anti-MBP and FITC-anti-GST, respectively. Three corresponding non-biotinylated proteins were also spotted onto the same slide, as controls, and the array was incubated with either individual antibodies (Figure 4.7), or a mixture of all three antibodies (Figure 4.8). Only specific binding between the biotinylated proteins and their corresponding antibodies were observed, regardless of the presence of other proteins (Figure 4.7) and antibodies (Figure 4.8), indicating the specific immobilization and versatility of this new protein array. Furthermore, no fluorescence signal was observed with the non-biotinylated control proteins (data not shown), confirming the essence of biotinylation for protein immobilization. 105 Chapter (a) (b) (c) Figure 4.7. Site-specific immobilization of proteins via avidin-biotin interaction. Three biotinylated proteins : (a) EGFP, (b) MBP and (c) GST were arrayed onto avidin functionalized slides and individually detected with Cy3-anti-EGFP (green), Cy5-antiMBP (red) and FITC-anti-GST (blue), respectively (a) (b) (c) Figure 4.8. Site-specific immobilization of proteins via avidin-biotin interaction. Three biotinylated proteins : (a) EGFP, (b) MBP and (c) GST were arrayed onto avidin functionalized slides and detected with a mixture of Cy3-anti-EGFP (green), Cy5-antiMBP (red) and FITC-anti-GST (blue) 4.2.3 Arrays of Functionally Active Proteins The most critical issue in generating a protein array is to ensure that proteins maintain their native activity, as it is previously known that proteins tend to denature on glass surfaces. In order to confirm that biotinylated proteins immobilized on the avidin slide retain their proper folding, the native fluorescence of EGFP on the slide was monitored 106 Chapter (Figure 4.9). No loss of fluorescence intensity was observed after prolonged incubation at 0C, suggesting that folding of the protein was properly maintained on the slide. Figure 4.9. Fluorescence from the native EGFP In a separate experiment, a slide immobilized with EGFP, MBP and GST was incubated with Cy3-labeled glutathione (Figure 4.10), a known natural ligand of GST. The result showed exclusive binding between GST and glutathione (Figure 4.11), further indicating full retention of the native GST activity. SH O O H N H2N N H OH O O OH Figure 4.10. Structure of glutathione 107 Chapter Figure 4.11. Functional activity of arrayed biotinylated GST. Biotinylated GST was arrayed onto avidin functionalized slides and incubated with its specific natural ligand, glutathione, labeled with Cy3. Furthermore, all data gathered thus far indicates that the presence of avidin as a molecular layer between the immobilized proteins and the glass surface also serves to minimize nonspecific absorption of proteins. Future improvement may be readily made by using streptavidin as the immobilization agent on the slide in place of avidin, which is a glycoprotein and known to have higher nonspecific binding characteristics.28 4.2.4 Comparison of Avidin-Biotin Interaction Stability with Other Existing SiteSpecific Immobilization Strategies Thus far, the only reported method for site-specific attachment of proteins in a microarray has been the immobilization of His-tag proteins on slides functionalized with Ni-NTA.12 However, the binding between His-tag proteins and Ni-NTA complex is not very strong, and incompatible with many commonly used chemicals such as DTT, SDS, EDTA, etc. The binding is also depleted outside the to 10 pH range, or when the buffer contains high concentrations of common salts. On the contrary, as mentioned in chapter 3, the binding between biotin and avidin is one of the strongest known non covalent interaction. Avidin is also extremely stable,16 108 Chapter making it an ideal agent for slide functionalization since glass slides can be prepared in advance and stored before being spotted. In addition, the interaction between avidin and biotin is instantaneous, hence requiring no incubation for protein immobilization. In order to confirm the benefit of avidin-biotin linkage, slides immobilized with GST were first subjected to a number of harsh washing conditions, and then detected with FITC-labeled anti-GST for any loss of GST on the surface. No loss of GST was observed even after the slide had been treated with 1M acetic acid at pH 3.3, or 60 0C water or even M GuHCl for prolonged time (Figure 4.12). For comparison, we have also prepared Ni-NTA slides according to published protocols.29 On the contrary to the avidin slides, when these GFP-spotted slides were treated with any of the above harsh conditions, the immobilization of the his-tag protein on the Ni-NTA was completely removed. (a) (b) (c) (d) Figure 4.12. Biotin avidin interactions subjected to harsh conditions. Biotinylated GST was arrayed on four avidin functionalized slides. Three slides were soaked in (a) acetic acid solution pH 3.3, (b) 60 ºC water, (c) M GuHCl and (d) no treatment for 30 min, and probed FITC-anti-GST. 4.3 Summary Findings described here present a new strategy for site-specific protein biotinylation and immobilization on a glass surface, generating a novel protein array on which 109 Chapter proteins are oriented optimally, and able to retain their native activity suitable for subsequent biological screenings. The advantage of avidin/biotin linkage over Histag/Ni-NTA strategies for protein immobilization is highlighted by its ability to withstand a variety of chemical conditions, which may make this new protein array compatible with most biological assays. 4.4 Materials and Methods 4.4.1 Chemicals All chemical were obtained from normal suppliers. The cysteine biotin was synthesized in Prof Yao’s lab, chemistry Department.30 The cloning of target genes into pTVB1 expression vector and on-column biotinylation were done by Rina Lue in Prof Yao’s lab, department of biological sciences, NUS.31 The sequence specific monoclonal antibodies, anti-EGFP and anti-MBP, were obtained from available commercial sources, and labeled with Cy3-NHS and Cy5NHS (Amersham Pharmacia, USA) respectively. The antibody was reacted with the dye for one hour in 0.1 M NaHCO3, pH 9, according to manufacturer’s protocols and purified with a NAP5 column (Amersham Pharmacia, USA). The anti-GST was purchased as a FITC-conjugate (Molecular Probes, USA). 4.4.2 Spotting of Biotinylated Proteins on Avidin Functionalized Slides and Fluorescent Antibody-Based Detection Glass slides were cleaned in a piranha solution and derivatized with a % solution of 3-glyicidoxypropyltrimethoxisilane (95 % ethanol, 16 mM acetic acid) for hr and 110 Chapter cured at 150 °C for hours. The epoxy slides were reacted with a solution of mg/mL avidin in 10 mM NaHCO3 for 30 minutes, washed with water, air dried, and the remaining epoxides were reacted with a solution of mM aspartic acid in a 0.5 M NaHCO3 buffer, pH 9. The biotinylated proteins were dissolved in PBS buffer, pH 7.4, and spotted onto the avidin functionalized slides using an ESI SMA arrayer (Toronto, Canada). No incubation was necessary before the slide were further processed by washing with PBS and drying in air. The spotted slides were incubated with the labeled antibody (or mixture of antibodies) for hour, washed times, each time for 15 with PBST (PBS + 0.1 % Tween 20), dried and scanned with an ArrayWoRx microarray scanner (Applied Precision, USA). 4.4.3 GST Assay The N-terminal amine group of glutathione was selectively labeled with Cy3-NHS by reacting the molecule overnight with the dye in sodium phosphate buffer at pH 7. The reaction was subsequently quenched with ethanolamine for 12 hours to degrade the remaining Cy3-NHS Biotinylated GST was arrayed on avidin slides. Following incubation with the Cy3-labeled glutathione for one hour, the slide was washed with PBST as described earlier, and the specific binding between GST and glutathione was visualized with an ArrayWoRx microarray scanner. 4.4.4. Treatment of Avidin Slides Under Harsh Conditions Biotinylated GST was arrayed on four avidin functionalized slides. Three slides were soaked in (1) 1M acetic acid solution at pH 3.3, (2) 60 ºC water, (3) M GuHCl and (4) no treatment for 30 min, and probed with FITC-anti-GST. For comparison, we 111 Chapter have also prepared Ni-NTA slides according to published protocols. Briefly, epoxy slides were incubated with NTA dissolved in NaHCO3. The slides were washed in water and soaked in 100 mM NiSO4 for at least hour, washed with 0.2 M acetic acid, 100 mM NaCl to give the Ni-NTA slides. GFP was expressed with a his-tag, and spotted it onto Ni-NTA slides. When this GFP-containing slide was treated with any of the above harsh conditions, the immobilization of the his-tag protein on the Ni-NTA was completely removed 4.5 References Gygi, S. P.; Rochon, Y.; Franza, R. B.; Aebersold, R. Mol. Cell. Biol. 1999, 19, 1720 Lee, K.-B.; Park, S.-J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702 MacBeath, G.; Koehler, A. N.; Schreiber, S. L. J. Am. Chem. Soc. 1999, 121, 7967 Hergenrother, P. 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Soc. 2000, 122, 5421 27 Tolbert, T.J.; Wong, C.-H. Angew. Chem. Intl. Ed. 2002, 41, 2171 28 Savage, D.; Mattson, G.; Nielander, G.; Morgensen, S.; Conklin, E. AvidinBiotin Chemistry: A Handbook, 2nd Ed.; Pierce Chemical Co. 29 Hochuli, Dobeli, Schacher, J. Chrom. A. 1987, 411, 177 30 Lesaicherre, M.-L.; Lue R. Y. P.; Chen, G. Y. J. ; Zhu, Q., Yao, S.Q. . J. Am. Chem. Soc. 2002, 124, 8768 31 “Expression and Purification of recombinant proteins with intein”, Honors Thesis, Lue Yee Peng, Rina, 2002, National University of Singapore 114 [...]... 4 proteins are oriented optimally, and able to retain their native activity suitable for subsequent biological screenings The advantage of avidin/biotin linkage over Histag/Ni-NTA strategies for protein immobilization is highlighted by its ability to withstand a variety of chemical conditions, which may make this new protein array compatible with most biological assays 4. 4 Materials and Methods 4. 4.1... (2) 60 ºC water, (3) 4 M GuHCl and (4) no treatment for 30 min, and probed with FITC-anti-GST For comparison, we 111 Chapter 4 have also prepared Ni-NTA slides according to published protocols Briefly, epoxy slides were incubated with NTA dissolved in NaHCO3 The slides were washed in water and soaked in 100 mM NiSO4 for at least 1 hour, washed with 0.2 M acetic acid, 100 mM NaCl to give the Ni-NTA slides... Nucleic Acids Res 1998, 26, 141 4 21 Sano, T.; Cantor, C R.; Proc Natl Acad Sci U.S.A 1990, 87, 142 22 Xu, M.-Q.; Evans, T C Methods 2001, 24, 257 23 Dawson, P.E.; Muir, T.W.; Clark-Lewis, I.; Kent, S.B.H Science 19 94, 266, 776 24 Evans, T C.; Benner, J.; Xu, M.-Q J Biol Chem 1999, 2 74, 3923 25 Muir, T W.; Sondhi, D.; Cole, P A Proc Natl Acad Sci U.S.A 1998, 95, 6705 113 Chapter 4 26 Tolbert, T.; Wong, C.-H... one hour in 0.1 M NaHCO3, pH 9, according to manufacturer’s protocols and purified with a NAP5 column (Amersham Pharmacia, USA) The anti-GST was purchased as a FITC-conjugate (Molecular Probes, USA) 4. 4.2 Spotting of Biotinylated Proteins on Avidin Functionalized Slides and Fluorescent Antibody -Based Detection Glass slides were cleaned in a piranha solution and derivatized with a 1 % solution of 3-glyicidoxypropyltrimethoxisilane... Biochem 1996, 2 34, 60 14 He, M.Y.; Taussig, M J Nucleic Acids Res 2001, 29, e73 15 Green, N M.; Toms, E J Biochem J 1973, 133, 687 16 Reznik, G O.; Vajda, S.; Cantor, C R.; Sano, T Bioconjugate Chem 2001, 12, 1000 17 Cull, M G.; Schatz, P J Methods Enzymol 2000, 26, 43 0 18 Cronan, J E.; Ree, K E Methods Enzymol 2000, 27, 44 0 19 Taki, M.; Sawata, S Y.; Taira, K J Biosci Bioeng 2001, 92, 149 20 Smith, P... SMA arrayer (Toronto, Canada) No incubation was necessary before the slide were further processed by washing with PBS and drying in air The spotted slides were incubated with the labeled antibody (or mixture of antibodies) for 1 hour, washed 4 times, each time for 15 min with PBST (PBS + 0.1 % Tween 20), dried and scanned with an ArrayWoRx microarray scanner (Applied Precision, USA) 4. 4.3 GST Assay... Soc 2000, 122, 542 1 27 Tolbert, T.J.; Wong, C.-H Angew Chem Intl Ed 2002, 41 , 2171 28 Savage, D.; Mattson, G.; Nielander, G.; Morgensen, S.; Conklin, E AvidinBiotin Chemistry: A Handbook, 2nd Ed.; Pierce Chemical Co 29 Hochuli, Dobeli, Schacher, J Chrom A 1987, 41 1, 177 30 Lesaicherre, M.-L.; Lue R Y P.; Chen, G Y J ; Zhu, Q., Yao, S.Q J Am Chem Soc 2002, 1 24, 8768 31 “Expression and Purification... Yao’s lab, chemistry Department.30 The cloning of target genes into pTVB1 expression vector and on-column biotinylation were done by Rina Lue in Prof Yao’s lab, department of biological sciences, NUS.31 The sequence specific monoclonal antibodies, anti-EGFP and anti-MBP, were obtained from available commercial sources, and labeled with Cy3-NHS and Cy5NHS (Amersham Pharmacia, USA) respectively The antibody... (c) (d) Figure 4. 12 Biotin avidin interactions subjected to harsh conditions Biotinylated GST was arrayed on four avidin functionalized slides Three slides were soaked in (a) acetic acid solution pH 3.3, (b) 60 ºC water, (c) 4 M GuHCl and (d) no treatment for 30 min, and probed FITC-anti-GST 4. 3 Summary Findings described here present a new strategy for site-specific protein biotinylation and immobilization...Chapter 4 Figure 4. 11 Functional activity of arrayed biotinylated GST Biotinylated GST was arrayed onto avidin functionalized slides and incubated with its specific natural ligand, glutathione, labeled with Cy3 Furthermore, all data gathered thus far indicates that the presence of avidin as a molecular layer between the immobilized proteins and the glass surface also serves to minimize nonspecific . Chapter 4 CHAPTER 4 INTEIN-MEDIATED BIOTINYLATION OF PROTEINS AND ITS APPLICATION IN A PROTEIN MICROARRAY 4. 1 Introduction 4. 1.1 Developing Microarrays of Functionally Active Proteins DNA microarray. by its ability to withstand a variety of chemical conditions, which may make this new protein array compatible with most biological assays. 4. 4 Materials and Methods 4. 4.1 Chemicals. water, (3) 4 M GuHCl and (4) no treatment for 30 min, and probed with FITC-anti-GST. For comparison, we 111 Chapter 4 112 have also prepared Ni-NTA slides according to published protocols.