recombinant protein protocols

1 Overview of Experimental Strategies on the Detection and Isolation of Recombinant Proteins and Their Applications Rocky S. Tuan 1. Introduction Recent advances m recombinant DNA technology have permitted the direct cloning of DNA fragments (either derived from naturally occurring or artifi- cially designed gene sequences) mto various cloning vectors mcludmg bacte- riophages, plasmids, and viruses. Such recombinant constructs represent the basic reagents of molecular biology. A major application utihzmg cloned DNA sequences is the expression of the cloned DNA mto a protein product, i.e., the expression of recombinant genes. Because the cloned DNA sequences may be modified or altered, recombinant expression technology thus enables the mvestigator to “custom-design” the final protem product Furthermore, most expression vectors are designed to allow the linking of various “tags” to the expressed recombinant protein to facilitate subsequent detection and isolation. This chapter provides a brief overview of the technologies currently employed m “taggmg” expressed recombinant protems and the corresponding detection and isolation methodologies, as well as some of the applications utilizing recombinant gene products. 2. Molecular Tags and Reporters The basic strategy m “labeling” or “tagging” a cloned sequence is to place either upstream or downstream a translationally m-frame sequence correspond- ing to a polypeptide domain or protein that exhibits highly active or distmct properties not found m the host cell. In this manner, the recombinant hybrid protein, containing the tag and the desired expressed gene product, may be detected and/or isolated on the basis of the unique properties of the tag. In From Methods m Molecular Bology, vol 63 Recombrnant Rote/n Protocols Detecftoon and /so/at/on Edlted by R Tuan Humana Press Inc , Totowa, NJ 3 4 Tuan some instances, an additional sequence correspondmg to a specific protease cleavage sate is Inserted between the tag and the cloned sequence, such that treatment of the final recombmant hybrid protein with the appropriate protease produces the desired gene product from the tag. Chapter 2 (Groskreutz and Schenborn) m this book provides further background for the general rationale used in constructmg an expression vector. 2. I. Enzymes Owmg to their ability to catalyze specific reactions yielding distmct, detect- able products, enzymes are probably the most popular molecular tag for expression of recombmant genes. The most commonly used enzymes include. chloramphenicol acetyltransferase (CAT), firefly luctferase; j3-galactosidase, alkalme phosphatase; and P-glucuromdase Some of the key reasons for select- mg these enzymes as functional labels include high signal-to-background ratios of the catalyzed reactions, high stability of enzyme activity, and the high sensi- tivity for detection A number of methods are currently m use for the detection of enzyme activity, mcludmg standard calorimetric assays, more sensitive fluo- rescence- or lummescence-based procedures, chromogenic histochemistry, and immunohistochemistry or solution-phase immunoassays such as radtoim- munoassay or enzyme-lmked immunosorbent assay (ELISA). The specific characteristics of some of these enzymes and then- respective detection pro- tocols are presented m detail m a number of chapters m this book (Chapters 3, 4, 5, and 6). 2.2. Ligand-Binding Labels Another type of molecular interaction that has been exploited to generate detectable activities m recombmant gene expression mcludes those mvolving specific, high-affimty ligand binding. In this manner, the recombmant product possesses the ability to interact with a specific hgand, which ideally is not a property of the host cell proteins. Using a labeled ligand, the correspondmg recombinant product may be clearly identified. Alternatively, another reagent, either a protein or a chemical (which is itself labeled), may be used to detect the bound ligand, and thus the recombmant protem. In many instances, the ligand may be immobilized onto a solid support, such as chromatography resins and gels, to develop affinity fractionation methods for isolation and purification of the desired recombmant product. Examples of these proto- cols may be found m a number of chapters m this book, dealing with spe- cific ltgand-binding entitles such as: maltose-bmdmg protein, which allows purification of the chimeric protein on amylose columns; Protein A, which recognizes the Fc domain of mnnunoglobulin G; streptavtdm, which binds with extremely high affinity and specificity to btotm, and hexahistidme peptide se- Overview 5 quence, which has hrgh metal affinity, i.e., applicable for affimty purification on nickel-mtrilotriacetate column. These topics are covered m detail m Chap- ters 9, 10, 11, and 12 2.3. Expression-Coupled Gene Activation Another means to detect recombinant gene expression, which has recently gamed substantial popularity, is the couplmg of recombinant gene expression to the transactivation of another unique gene This approach, an example of which is the yeast two-hybrid system, is particularly useful for the detection of interacting proteins, and the assay is performed m vivo rather than m vitro, thus permittmg the detection of such proteins m their native, biologically active state. The yeast two-hybrid system takes advantage of the fact that many eukaryotic transcrtption activators are made up of structurally separable and functionally independent domains. For example, the yeast transcriptional acti- vator protein GAL4 contams a DNA-binding domain (DNA-BD), which rec- ognizes a 17 base-pan DNA sequence, and an acttvation domain (AD). Upon DNA-BD binding to the specific upstream region of GAL4-responsive genes, the AD interacts with other components of the machinery to initiate transcrip- tion. Thus, both domams are needed m an mteractive manner for specific gene activation to take place. In the popular yeast two-hybrid system, the two GAL4 domains are separately fused to genes encoding proteins that interact with each other, and these recombinant hybrid proteins are expressed in yeast. Interac- tion of the two-hybrid proteins brings the two GAL4 domains m close enough proximity to form a functional gene acttvator, resultmg m the expression of specific reporter gene(s), thereby rendering the protein interaction, 1.e , expres- sion of the desired recombinant protein, phenotypically identifiable. In prac- tice, the target protein gene is ligated to the DNA-BD m the form of an expression vector. The gene of interest, whose acttvtty Includes mteraction with the target protein, IS ligated into an AD vector. The two hybrid plasmids are then cotransformed mto specialized yeast reporter strain. Expression of the desired gene thus activates a known GAL4 responsive gene(s) and confers spe- cific phenotype to the host cell, which can be selectively identified Protocols utilizing the two-hybrid system and its variants are described m several chap- ters m this book (Chapter 12, 15, and 16). 2.4. lmmunospecific Detection Another type of recombinant label or tag consists of components to which specific antibodies are available In thts manner, mmmnoassays and nnmuno- affinity chromatography may be used efficiently to detect and isolate, respec- tively, the recombinant protem. Momand and Sepehrma (Chapter 14) illustrate how this prmciple may be exploited using recombinant p53 as an example, and 6 Tuan Olesen et al. (Chapter 7) present methodologres based on chemrlummescent rmmunoassays using enzyme-conjugated secondary antibodies. Recombinant protem expressing cells may also be cloned and detected by tmmunodetectron methods as described by Gibson et al. (Chapter 8). 2.5. Expression-Coupled Alteration of Cellular Activity The rdentificatton and clonmg of multrdrug resistance genes such as MDRl, which is responsible for the simultaneous resistance of cells to multtple struc- turally and functionally unrelated cytotoxtc agents, offers the potential to use such genes m fusion gene constructs for the purpose of detection and isolatron of the expressing cellular clone. Germann (Chapter 13) describes such an application pertaining to P-glycoprotem. 2.6. Labels with Unique Chemical Characteristics The green fluorescent protein (GFP) of the jellyfish, Aequora victona, fluo- resces following the transfer of energy from the Ca2+-actrvated photoprotein, aequorin. GFP has been cloned, and, when expressed in prokaryotic and eukaryottc cells, yields green fluorescence when exctted by blue or ultravtolet light. Recent developments have focused on utrlizing the GFP as a reporter gene to permit the detection of recombinant gene expression m vlvo (see below). 3. Detection of Gene Expression The expression of spectfic genes m a recombinant form consisting of cht- merit or hybrid labels has greatly facrlitated then detection. A number of chap- ters m this book (Chapters 18, 19, 20, 21, 22, 23, and 24) focus on recent developments in the detection of gene expression zn situ, utilizing DNA or RNA probes, as well as PCR amplificatron. Smce zn sztu hybridizatron, in sztu PCR, and label-spectfic htstochemrstry (e.g., P-galactosrdase hlstochemrstry), utrlizmg tissue sectron or whole-mount tissues and embryos, yield mformatron on gene expression at the native, indrvtdual cell and tissue level, they are pow- erful techniques in gammg mformation on the functtonal aspects of gene expression. In particular, in transgemc expertmentatrons, where a transgene IS introduced into and expressed in an animal, the abtlrty to correlate gene expression and altered cell/tissue phenotype allows the investigator to directly assess the function of the gene of interest. Examples of recombmant label- specific detection protocols include those based on P-galactosidase (Chapter 18) and the fluorescent jellyfish GFP (Chapter 24). In situ hybridization IS based on hybrrdrzatron to spectfic mRNA sequences by labeled DNA and RNA probes (Chapters 19,20,2 1, and 22), whereas sequence-specific amplrficatton by in situ PCR provides both gene expression detection and cloning possrbrlr- ties (Chapter 23). By coupling immunohrstochemrstry with zn sztu hybridlza- Overview 7 tion, it IS posstble to examme gene expression at both the protein and mRNA levels (Chapters 19 and 22). 4. Applications of Recombinant Gene Expression The productron of recombinant gene products IS one of the maJor success stories of modern molecular biology. This book samples some of the applrca- trons to Illustrate the present state and future potentral of such a technology. Thus, m addmon to answering fundamental questions related to regulation of gene expression, gene structure and function, and other basic issues of molecu- lar biology, the technology of recombinant genes has revolutionized modern brotechnology and bromedicme. For example, gene therapy, which arms to compensate for gene defects and/or deliver therapeutrc gene products for spe- c~fic diseases resultmg from defective genes, is crrtically and totally dependent on the design of expression and delivery vectors, which permit targeted and regulated expression of the cloned gene(s) (see Chapters 28, 29, 30, and 3 1). On the other hand, recombinant gene technology also makes rt possible to cus- tom-desrgn gene sequences to “blosyntheslze” novel bropolymers of unique physrcochemical properties (see Chapter 27), which may be used for apphca- trons m biomaterral, pharmaceutical, agricultural, and other mdustrres Finally, it should be noted that the current state of recombmant technologtes IS capable of utibzmg a wade spectrum of manufacturing umts (the “btoreactors”) for the custom-designed protein products, mcludmg E. colz (Chapter 26), yeast, cul- tured cells, and transgenic animals (Chapter 25 j. [...]... mammalian and bacterial GUS proteins can be distmgmshed from each other by then differing pH optima In addition to the uses of GUS as a genetic reporter, GUS fusion proteins can be created to study protein transport and localization in both plant and animal cells (76) 4.5 Human Growth Hormone (hGH) 4.5.1 Origin of Reporter Gene Human growth hormone gene 4.5.2 Protein Characterisbcs The protein is 24-25,000... greenfluorescent protein Bzochemlstry 32, 12 12-12 18 Chalfie, M , Tu, Y., Eusktrchen, G , Ward, W W , and Prasher, D C (1994) Green morescent protein as a marker for gene expresston Science 263,802-805 Wang, S and Hazelrtgg, T (1994) Imphcattons for bed mRNA localization from spatial distribution of exu protein m Drosophila oogenests Nature 369,400-403 3 Detection of Recombinant Protein Based on Reporter... Gene Transposon 9 of E coli (53) 4.1.2 Protein Characterrstics CAT is a trimertc protein comprtsing three ldentrcal subunits of 25,000 Dalton (54) The CAT protein is relattvely stable m the context of mammalian cells, although the mRNA has a relatively short half-hfe, makmg the CAT reporter especially suited for transtent assaysdesigned to assessaccumulation of protein expression (55) 4.7.3 Enzymatic... expressing a transacting protein or RNA of interest The protein could be a regulatory transcription factor that binds to the promoter region of interest, cloned upstream of the reporter gene For example, when tat protein is expressed from one vector in a transfected cell, the activity of the HIV-LTR linked to a reporter gene increases, and is reflected m the increase of reporter gene protein activity (35)... secreted reporter protein, low endogenous reporter background, and a wide variety of easy and sensittve assaysmake SEAP a convenient and versatile reporter system.The assays for SEAP activity are identical to those described for AP 25 Reporter Systems 4.8 Green Fluorescent Protein (GFP) 4.8.1 Origin of Reporter Gene The green fluorescent protein from the jellyfish, Aequorea vzctoraa 4.8.2 Protein Characteristics... screening (3 7) 3.2 Men tifica tion of Interacting Proteins Interacting pairs of proteins can be identified in vwo using a clever system developed by Stanley Fields and coworkers (38,39) Known as the two-hybrid system, the interacting proteins of interest are brought together as fusion partners-one is fused with a specific DNA binding domain and the other protein is fused with a transcrtptional activation... Light-stimulated GFP fluoresces in the absence of any other proteins, substrates,or cofactors Therefore, unltke any of the other available reporters, GFP gene expression and locahzatton can be momtored m hvmg organisms and m live or fixed cells using only UV or blue-light tllumination (87,881 Additlonally, GFP fusion proteins can be constructed to study protein transport and locahzatron (88) Other advantages... for proteins that interact with a protein of interest Proc Nat1 Acad Scz USA 88, 9578-9582 39 Fields, S and Song, 0 (1989) A novel genetic systme to detect protem-protem mteractions Nature 340,245-246 40 Fearon, E R., Finkel, T , Gilhson, M L , Kennedy, S P., Casella, J F., Tomaselh, G F , Morrow, J S , and Van-Dang, C (1992) Karyoplasmic interaction selection strategy a general strategy to detect protein- protein... the reporter is required by measuring the reporter mRNA or protein Detection of the mRNA is a more direct measure of reporter gene expression than protein detection since the effects of transcription are observed directly, avoiding possible artifacts that may be the result of downstream processmg events such as Reporter Systems 17 translation or protein instability Reporter mRNA can be detected by Northern... cells whtch have not been transfected, as compartsons for the cell-free and znsztuanalyses 4.4 j3-Glucuronidase (GUS) 4.4.1 Origin of Reporter Gene The gus A gene from E coil 4.4.2 Protein Characteristics A tetrameric glycoprotein composed of four identical subunits of 68,000 Dalton is localized predominantly to the acidic environment of the lysosomes 4.4 3 Enzymatic Reaction GUS 1san exoglycosidase . separately fused to genes encoding proteins that interact with each other, and these recombinant hybrid proteins are expressed in yeast. Interac- tion of the two-hybrid proteins brings the two GAL4. gene(s), thereby rendering the protein interaction, 1.e , expres- sion of the desired recombinant protein, phenotypically identifiable. In prac- tice, the target protein gene is ligated to the. ing to a polypeptide domain or protein that exhibits highly active or distmct properties not found m the host cell. In this manner, the recombinant hybrid protein, containing the tag and the