Show Me The Money: Prokaryotic Expression Vectors And Purification Systems

Face it, you're an agent! Scientists should really consider themselves professional promoters-promoters not too different from the type portrayed in a not-to-be-mentioned recent movie (it made enough money). Our clients are not jocks looking for some franchise to pick up a multi-year contract; our clients are genes that have biologically-athletic capabilities. Consider that genes are not just average run-of-the-mill sequences playing weekend pick-up games whenever they get enough friends together. They have reached "professional status" as the result of being highly evolved to respond efficiently and perform incredibly specialized functions. Proteins have achieved things, have accomplished things, that are difficult for us to reverse-engineer even a poor version of. These "professional" proteins have talent and years of experience that allow them to play in the big leagues. Luckily there are enough genes around for everyone to represent! For most genes, proteins are the athletic embodiment of these professional activities. Our task as agents then, is to find the correct medium for these proteins to "show their stuff". Occasionally, we give them advice (mutations) and hopefully improve their game-sometimes. As an agent, your first responsibility is to identify the league for which your particular client (protein) is best suited. There are, of course, the standard prokaryotic, yeast and mammalian leagues. Within the last few years, a number of expansion leagues have been added, including Baculovirus and specialized spin-offs such as viral systems. Beginning with this issue, we will take a look at expression vectors available for prokaryotic systems, particularly E. coli. This series will continue in later issues and will take a look at what the other franchises are up to. As an expression system, bacteria have some good features as well as a few that can create headaches for your client. The clearest advantages of bacterial systems are those of cost, ease of manipulating sequences, transforming, and producing proteins. The features that result in headaches are generally related to the fact that some recombinantly expressed proteins can have deleterious effects on the host strain during growth phases. Lab life becomes more complicated at times because some nonbacterial proteins are insoluble when overexpressed in E. coli. In addition, many proteins are translationally processed or modified in their natural hosts, but not in bacterial systems; these modifications usually affect folding activities and biological functionality. It seems, therefore, a matter of luck to express a protein and retain functional activity. Despite these caveats, bacterial systems are a good place to start to assure reading frames or for the production of protein for antisera preparation. Luckily there are a number of clever bacterial expression systems that help circumvent these potential headaches and put you and your client on the fast track. More importantly, these systems are coupled with supporting materials and reagents in kit formats that can help you clone, express, and purify recombinant proteins with a fair amount of ease. Promoters are DNA sequences to which the endogenous bacterial RNA polymerase will bind to initiate transcription. The promoter has an important function in that it determines the polarity of the transcript by specifying which strand will be transcribed. More important, how tightly regulated a promoter/operator is will greatly affect the ability to express proteins. Most bacterial promoters used in expression vectors consist of two elements located (-35) and (-10) from the actual transcriptional start. These elements comprise the consensus sequences that are bound by a specific transcription factor and the RNA polymerase. One of the most commonly encountered promoters is the trc promoter. DNA sequences that act in conjunction with promoters and bind repressor molecules regulate the induction of transcription. One of the most commonly used in E. coli is the lacO/ lacIq repressor system. In this system, transcription is virtually shut off until the promoter is derepressed by the addition of IPTG. At this point, the promoter is freed (i.e. the repressor no longer physically blocks transcription) and transcription is turned on. Inducible elements provide the ability to keep expression of the target gene off, should it produce a product that might be toxic to the host strain. There are a number of different methods of regulation that are available in commercial expression systems. While most are capable of inducing tremendous levels of expression, even slightly leaky expression during culture expansion can limit final product yield. One recently introduced promoter system, the arabinose-regulated promoter of the pBAD (Invitrogen) family of vectors, allows dose-dependent expression of target proteins. Arabinose-Regulated Promoter (Invitrogen pBAD Vector): This system generates very low levels of uninduced expression through glucose-catabolite-dependent repression. The pBAD promoter is induced by arabinose. Adding arabinose into the medium in increasing concentrations induces transcription in a dose-dependent manner. T7 Expression Systems (Novagen, Promega, Stratagene): The pET-based vectors utilize the T7 RNA polymerase-based expression vector of Moffat and Studier, J. Mol. Biol,. 189: 113-130, (1986) to achieve very high levels of protein expression. The power of the pET system is that the T7 RNA polymerase is specific for its own promoter, which is found only on the expression plasmid. During growth and culture expansion, the T7 promoter is under the tight control of the lacIq gene, repressing any expression that might adversely affect bacterial growth. When induced with IPTG, rather than directly inducing the T7 promoter 5' to the target gene, a T7 RNA polymerase is expressed in host E. coli, allowing transcription from the T7 promoter. Transcription and translation can be accomplished in only a few hours, with the expressed protein often comprising the most abundant cellular component. Trc/Tac Promoter Systems (Clontech, Invitrogen, Kodak, Life Technologies, MBI Fermentas, New England BioLabs, Pharmacia Biotech, Promega): Trc promoters are IPTG-inducible hybrid promoters. The Trc promoter is a trp/lac fusion, where the (-35) position is derived from trp and the (-10) position is obtained from lacUV5 promoter elements. While extremely strong, some low-level expression of the recombinant protein may occur during growth. PL Promoters (Invitrogen pLEX and pTrxFus Vectors): PL-based systems place the protein of interest under the tight transcriptional control of the Lambda cI repressor protein. The repressor protein must be engineered into the E. coli host or be incorporated into the vector itself. The repressor protein is placed under the control of an inducible promoter. Expression of the cI repressor binds to the operator of the PL promoter to prevent transcription of the recombinant gene. Induction occurs with the addition of tryptophan, which prevents expression of the repressor, allowing transcription for the PL promoter. Lambda PR Promoter (Pharmacia pRIT2T Vector): The PR promoter of the bacteriophage Lambda provides for thermo-inducible expression of proteins when temperatures are shifted during growth from 37°C to 42°C. Induction is accomplished in 90 minutes. Phage T5 Promoter (QIAGEN): The phage T5 promoter provides a strong recognition site for E. coli RNA polymerase and can direct the expression of targets to levels up to 50% of total cellular protein. The promoter is regulated by two lactose operator elements. tetA Promoter (Biometra pASK75 Vector): The tetA promoter is induced by the addition of anhydrotetracycline in concentrations that are not antibiotically effective. This promoter is not regulated by any endogenous cellular mechanisms and therefore is not influenced by catabolic repression. Stabilizing and optimizing elements; ribosome binding sites, transcriptional and translational termination sequences In prokaryotic systems, anti-termination elements are incorporated into vectors to stabilize the RNA polymerase on the DNA template. These elements help ensure optimal transcript elongation during message synthesis. Transcription terminators are used to signal the active RNA polymerase to release the DNA template and halt transcription of the newly transcribed RNA. These terminators are ordinarily positioned downstream of the multicloning site and act to prevent pausing, prevent pre-mature termination, and limit read-through of transcription, which adversely affects plasmid replication. Use of transcription terminators such as rrnB T1 and T2 from E. coli 5S rRNA are especially important for use with strong promoters. Ribosome binding sites (RBS) are small, open reading frames upstream of the coding sequence of interest engineered to encourage ribosomes to bind and translate the sequence of interest. An RBS (Shine-Dalgarno sequence) is required just upstream of the translational start to provide the context for efficient translation initiation. RBS sequences are engineered into vectors to enhance and stabilize ribosome binding. Often RBS elements may be "borrowed" from different sources or are the native RBS for the fusion species. Frequently used RBS elements are obtained from either the lacZ gene or the gene 10 of the bacteriophage T7. Many vectors are now engineered with DNA sequences encoding a specific peptide that, when fused to the expressed protein, may be used for one-step purification of the recombinant protein by high-affinity binding. One of the most popular systems is the 6xHis system in which six histidine residues enable the tagged-recombinant protein to be purified by a nickel-chelating resin. Often, an endopeptidase recognition sequence is also engineered between the affinity tag and the protein of interest to allow subsequent removal of the leader sequence, peptide-tag, or fusion sequences by enzymatic digestion. Many of the tags have functions beyond purification, with the distinction between fusion and tag becoming quite blurred. Some traditional tags pull double duty for purification as well as stabilization of the expressed protein. With the generation of antibodies, a fusion initially incorporated to stabilize expressed proteins may suddenly become a tag as well. Deciding which affinity purification method to use involves several issues including cost, position of the tag or fusion in the target, whether the final use necessitates removal of the tag, and the scale of the project. Additionally, important considerations are downstream purification strategies and the conditions that the target protein will experience when it is time to remove it from the affinity matrix. While many of the matrices are convenient and effective, cost can become a factor. Luckily, many of the matrices can be regenerated and used for additional rounds of purification. These basic decisions are also balanced against the limitations of expressing foreign sequences in a bacterial system. Insolubility of proteins tends to limit subsequent use of some expressed proteins. Fusions that assist in stabilizing expressed proteins are quite helpful in obtaining useful sources of recombinant proteins. Calmodulin-Binding Peptide (CBP) Tag (Stratagene pCAL Vectors): pCAL Expression Vectors contain a sequence encoding a CBP. The CBP tag allows the hybrid recombinant protein to bind to a calmodulin resin in the presence of low concentrations of calcium. Elution is accomplished in the presence of 2mM EGTA and neutral pH. The conditions are milder than in other tag systems. The CAP tag is one of the smaller tags, encoding a 4kDa tag. Smaller tags have potentially less impact on the protein of interest than larger tags. Glutathione S-Transferase (GST) Tag (Pharmacia pGEX Vectors): Vectors containing the GST-fusion tag allow encoded proteins to be efficiently purified from bacterial lysates utilizing an affinity matrix containing glutathione. Elution of the purified protein is accomplished under mild, nondenaturing conditions. The GST fusion adds a 26kDa tag to the recombinant protein, which can be removed when an endopeptidase cleavage site sequence is incorporated between the tag and the protein. 6xHIS Tag (Invitrogen, Life Technologies, Novagen, QIAGEN): 6xHis-tagging vectors fuse a six histidine-peptide to the recombinant protein. This small addition rarely affects protein structure to a significant degree and therefore usually does not require removal following purification of the protein. The 6-His residues impart a remarkable affinity for matrices containing nickel. The fact that binding can occur under native as well as under denaturing conditions distinguishes this affinity purification method from the others. Recombinant proteins, frequently encountered in inclusion bodies in bacterial expression systems, can be solubilized under denaturing conditions using urea or guanidine hydrochloride. The solubilized 6xHis-tagged proteins can then be purified by binding to nickel ions on the matrix. The strong affinity of the 6xHis tag tolerates denaturing conditions that facilitate the removal of nonspecific contaminants often associated with recombinant proteins expressed in bacteria. Elution is accomplished under mild conditions by either reducing the pH or adding imidazole as a competitor. and Companion Products Invitrogen ThioFusion Expression System His-Patch ThiolFusion System Anti-Thio Antibody ThioBond Resin ProBond Metal Binding Resin PL Expression System Xpress System Kodak E.coli Amino Terminal FLAG Expression Kit; E.coli Carboxyl Terminal FLAG Expression Kit Anti-FLAG Affinity Gel Anti-FLAG Monoclonal Antibodies Life Technologies ProEX HT Prokaryotic Expression System TEV Protease, recombinant New England BioLab IMPACTTM 1 System pMAL System Novagen pET Expression Systems S-Tag Rapid Assay Kit S-Tag AP Western Blot Kit S-Tag SRP Chemiluminescent Western Blot Kit S-Tag AP Chemiluminescent Western Blot Kit S-Tag Thrombin Purification Kit; S-Tag rEK Purification Kit Thrombin Cleavage Capture Kit Enterokinase Cleavage Capture Kit T7-Tag Antibody T7-Tag Affinity Purification Kit; CBD-Tag Purification Reagents and Kits HSV-Tag Antibody Pierce React-Bind Glutathione Coated Plates Immobilized Glutathione Immobilized GST ImmunoPure¨ Anti-Glutathione S-Transferase Reacti-Bind Dextrin Coated Plates Immobilized Dextrin-10 Reacti-Bind NeutrAvidinª Coated Plates ImmunoPure Goat Anti-Biotin Reacti-Bind Metal chelate Plates Pharmacia Bulk GST Purification Module RediPack GST Purification Module GST Purificaion Module Glutathione Sepharose PreScission Protease Promega PinPoint Protein Purification System ClipTM Fusion Protein Cleavage System PinPoint System and Clip System SoftLink Soft Release Avidin Resin TetraLink Tetrameric Avidin Resin QIAGEN QIAexpress Kit QIAexpress Sequencing Primers Ni-NTA Afarose Ni-NTA Superflow Ni-NTA Silica Ni-NTA Spin Kits Ni-NTA Spin Columns HIS Antibody Ni-NTA AP Conjugate Ni-NTA HRP Conjugate Ni-NTAHisSorb Srips Stratagene pET Expresion Systems Affinity Protein Expression and Purification System Affinity LIC Cloning Kit Affinity LIC Cloning and Protein Purification Kit Calmodulin Affinity Resin Affinity CBP Fusion Detection Kit Thomas F Unger can be contacted at tfunger@aol.com Dihydrofolate Reductase (QIAGEN): Short peptide sequences from the murine DHFR are fused to target proteins to increase stability of the target protein, as well as enhance its antigenicity. Thioredoxin Fusion Sequences (Invitrogen, pTrxFUS and pThioHis Vectors, Novagen pET-32 Vectors): Designed to maximize the accumulation of fusion proteins in the soluble fraction, a number of additional advantages are imparted when recombinant proteins are fused with sequences encoding thioredoxin. Fusion with thioredoxin helps overcome insolubility encountered with some bacterial systems. Additionally, in E. coli, thioredoxin accumulates in adhesion zones, which can be selectively released by rapid osmotic shock. The temperature stability of thioredoxin also allows temperatures as high as 80¡C to be tolerated during purification. These features can be exploited to simplify purification of expressed fusion proteins. Finally, thioredoxin possesses a unique active dithiol, which directs high-affinity binding to phenylarsine oxide, allowing rapid purification of expressed proteins. Protein A (Pharmacia pEZZ 18 and pRIT2T): The ability of Protein A to bind to IgG immunoglobulins continues to be a workhorse in biotechnology. pEZZ 18 contains two synthetic "Z" domains of the "B" IgG binding domains of Protein A and pRIT2T contains the natural IgG binding domains of protein A. Fusion proteins are easily purified using IgG Sepharose resins. The two Z domains add a 14kDa peptide to the recombinant protein. Biotinylation (Promega PinPointTM Vector): Biotinylation-based epitopes encode peptide sequences that become biotinylated in vivo during expression. Relying on the strong affinity of streptavidin for biotin, the biotinylated peptide acts as a purification tag for the fusion protein. Promega provides a unique monomeric avidin which allows elution of the fused proteins under mild non-denaturing conditions (5mM biotin), not possible with native avidin. Cellulose Binding Domain (CBD) (Novagen, pET CBD Vectors): CBD's allow for the purification of expressed proteins with cellulose or chitin matrices. These materials are generally very low cost and quite stable and may be found in a variety of forms including beads, powders, fibers, membranes, filters, and sheets. Additionally, the CBD fusion often increases thermostability of the recombinant protein, which may be useful in purification strategies or in simply increasing the stability of expressed proteins. Maltose Binding Protein (MBP) (New England BioLabs, pMAL Vectors): The malE gene encodes the MBP, which has the capability to bind tightly to amylose. The MBP is separated from the desired expressed sequence by a polylinker encoding ten asparagine residues. The linker is designed to assure the MBP can adequately bind amylose, which is immobilized on resin. In addition to aiding in expression and purification, the MBP fusion partner also helps keep proteins soluble and provides a choice of folding pathways. S-Peptide Tag (Novagen, selected pET Vectors): The 15 amino acid peptide (S-Tag) encoded by these vectors is a small, hydrophilic tag that has a strong affinity (Kd=109M) for a 104 amino acid protein (S-protein) derived from pancreatic ribonuclease A. When associated, the S-protein:S-peptide complex possesses ribonuclease activity (ribonuclease S). This strong association and activity have been exploited to measure S-Tag fusion protein (ribonuclease assay, which can detect as little as 5 fmol target) and can be used with reagents and resins for detection and purification purposes. For example, the S-Protein itself can be directly conjugated to alkaline phosphatase or horseradish peroxidase and used as a probe to detect S-Tag fusion protein by Western blot. Strep-tag (Biometra pASK75 Vector) A C-terminal fusion tag, the Strep-tag encodes a ten amino acid sequence which binds streptavidin. Affinity of the tag for streptavidin allows purification of recombinant proteins under mild conditions. Along with affinity-purification applications, the strep-tag can be utilized to directly detect recombinant proteins by Western blot or ELISA assays using streptavidin-enzyme (alkaline phosphatase) conjugates. Intein Mediated Purification with Affinity Chitin-Binding Tag (New England BioLabs, pCYB Vectors/IMPACT System): This chimeric system produces a C-terminally tagged protein in which a small 5kDa chitin-binding domain (CBD) from Bacillus circulans is physically separated from the target protein by the protein splicing element derived from the Saccharomyces cerevisiae VMA gene 1. The splicing element, or Intein, has been modified such that it undergoes a self-cleavage reaction at its N-terminus at low temperatures in the presence of thiols. The fusion protein is purified by passing extracts through a chitin column; the purified protein is then induced to undergo the Intein-mediated self-cleavage on the column by overnight incubation at 4¡C in the presence of DTT or b-mercaptoethanol. The target protein without additional residues is released while the chitin-binding fusion domain remains adhered to the resin. Figure reprinted with permission from New England Bio-Labs Immuno-reactive Epitopes (Invitrogen, Novagen, Kodak): While many vectors utilize fusion or epitope tags for multifunctional purposes, some vectors encode fragments that are essentially immunoreactive epitopes. Immunoreactive tags provide a rapid means of detecting tag-hybrid proteins with high specificity and affinity without needing to generate specific antisera to each protein of interest. The monoclonal anti-myc antibody detects recombinant proteins containing the myc epitope (GluGlnLysLeuIleSerGluGluAspLeuAsn). An alternative is the 11 amino acid epitope tag (GlnProGluLeuAlaProGluAspProGluAsp) derived from Herpes Simplex (HSV) glycoprotein D, which can be recognized by an anti-HSV monoclonal antibody. Kinase Sequences for in vitro Labeling (Stratagene, Pharmacia): To enable rapid in vitro labeling of proteins with 32P, some vectors incorporate the recognition sequence for the catalytic subunit of cAMP-dependent protein kinase as a fusion epitope. The site encoding the sequence (ArgArgAlaSerVal) is usually located between a primary fusion sequence and the sequence for the gene of interest. Expressed proteins can be directly labeled using protein kinase and [gamma 32P]-ATP (W.G. Kelin et al., Cell, 70, 351 1992.) Protein A Signal Sequence (Pharmacia pEZZ18): The protein A signal sequence of this vector results in the secretion of the expressed protein into the aqueous culture. ompT and ompA Leader Signal Peptides (Biometra, New England BioLabs, Kodak): The ompT and ompA leader sequence directs secretion of proteins into the periplasmic space. This periplasmic localization can simplify recovery of expressed proteins since osmotic shock treatments can enrich for proteins localized within the periplasm. malE Signal Sequence (New England BioLabs pMAL-p2): The normal malE signal sequence contains residues that direct the expressed fusion protein through the cytoplasmic membrane to the periplasm. This provides an alternate folding pathway that particularly helps in the formation of disulfide bonds. T7 gene 10 Leader Peptide (Novagen, Stratagene, Promega, Invitrogen): An 11 amino acid leader peptide (MetAlaSerMetThrGlyGlyGlnGlnMetGly) derived from the T7 major capsid protein (gene 10) is incorporated into vectors to provide an ATG source for fused proteins. This leader peptide is often used in T7 expression systems. Antibodies generated against this epitope can be used to detect the production of expressed protein in Western or immunoprecipitation assays. Cleaving the target protein from the fusion product Production of tagged or fusion proteins allows for rapid quantitation and purification of recombinant proteins, yet they have one serious flaw: the addition of the vector-derived epitope. While some epitopes such as the 6X His Tag are small and may be inconsequential, others are significantly larger and may affect the downstream use of the recombinant protein. By incorporating site-specific protease cleavage sites between the epitope/fusion and the target protein, these sequences can be removed. Keep in mind, however, that there is a possibility that the recombinant protein may have a proteolytic cleavage site within its own sequence or that many times, the protease may not adequately recognize its target site because of structural problems. Various methods of processing purified protein products are available. Consider, however, that in scale, there may be considerable expense incurred. Additionally, using a protease to mediate cleavage eventually requires that the protease itself must be removed following the reaction, in order to obtain a truly purified recombinant protein. To address this issue, some manufacturers have incorporated novel methods of releasing the target protein from the fusion product while simultaneously including a strategy of immobilizing the protease on a resin through use of an engineered tag on a recombinant endopeptidase. The PreScissionª Protease from Pharmacia Biotech is a genetically engineered fusion protein consisting of the 3C protease of the human rhinovirus type 14 and glutathione S-tranferase. This protease can be used either following purification of the recombinant protein, or while the target protein is bound to glutathione sepharose. Since the protease itself contains a GST tag, it remains bound to the sepharose matrix allowing rapid purification of the target away from the protease. New England BioLabs has recently introduced a vector that relies on Intein-mediated self-cleavage rather than proteolytic cleavage of the fusion. The Intein sequence mediates self-cleavage between the tag-epitope and the target sequence and liberates the target protein from the fusion species without the introduction of additional proteins. The affinity tag can remain immobilized, and purification of the target is relatively easy. Some commonly encountered protease/cleavage sites are: Thrombin (KeyValProArg/GlySer) Factor Xa Protease (IleGluGlyArg) Enterokinase (AspAspAspAspLys) rTEV (GluAsnLeuTyrPheGln/Gly): rTEV is a recombinant endopeptidase from the Tobacco Etch Virus. Intein-mediated self-cleavage (New England BioLabs) 3C Human rhino virus Protease (Pharmacia Biotech) (LeuGluValLeuPhe Gln/GlyPro) Polylinkers or MCSs are synthetic DNA sequences encoding a series of restriction endonuclease recognition sites that are engineered for convenient cloning of DNA into a vector at a specific position. MCS sites can range in size from two restriction sites and up. To accommodate reading frame differences with translational start sequences, many vectors are offered in an "A, B, C" format, meaning there is a single base shift between each vector, allowing easy cloning into each of the three reading frames. Additionally, some vectors are provided where the MCSs are in opposite orientations. For sequences where the amino-terminus has not been precisely defined, Kodak has developed a FLAG-Shift vector. These vectors contain a "shift" sequence that allows expression of an open reading frame without regard to the reading frame in which it was originally cloned. This shift is facilitated by ribosomal slippage caused by a run of A/Ts. LIC Vectors (ligation-independent cloning, Novagen, Stratagene) allow directional cloning of PCR products without restriction digestion or ligation reactions of the amplified products. LIC Vectors rely on sequence complimentarity between the primer used for amplification and the ends of the vector. These overhangs, following treatment with T4 polymerase, allow specific and efficient annealing and subsequent transformation directly into bacteria. Primers for the overhang can also be engineered to accomplish additional tasks. Novagen, for example, uses primers that encode the recognition site for Enterokinase. All vectors must contain a DNA sequence that directs binding of DNA polymerase and associated factors in order to maintain copies of the vector. Most vectors utilize elements from pBR322. Many vectors also provide an M13 origin of DNA replication (f1) allowing the production of single-stranded DNA for sequencing and mutagenesis. All vectors contain sequences that provide a means to select only those cells containing a vector. The following markers function by either conveying drug resistance on the host or enabling the host to compensate for the absence of an essential component in the media (auxotrophic markers): Ampicillin: interferes with a terminal reaction in bacterial cell wall synthesis. The resistance gene (bla) encoding beta-lactamase cleaves the beta-lactam ring of ampicillin. Tetracycline: prevents bacterial protein synthesis by binding to the 30S ribosomal subunit. The resistance gene (tet) specifies a protein that modifies the bacterial membrane and prevents transport of the antibiotic into the cell. Kanamycin: binds to the 70S ribosomes and causes misreading of messenger RNA. The resistant gene (Km) modifies the antibiotic and prevents interaction with the ribosome. With the ever-increasing rate of discovering new genes comes the equal challenge of understanding how the products of these genes are used and regulated, when they appear, and how they interact within cells. While much attention has been given to high-throughput PCR and sequencing, numerous advancements have been made in lower profile areas such as developing tools to rapidly express and purify proteins. Your decision regarding which expression format is best for a particular gene is similar to that made by a sports agent in deciding which team is the best fit for an athlete. For the latter decision, an agent needs to find a team where the athlete can make the best contribution. That decision is made by finding a team that allows the capabilities of the athlete to be fully realized. An all-star playing on a bad team is still talented, but one questions how good that athlete could be if given a better opportunity. This situation is analogous with bacterial expression vectors and companion products. Each protein will have some unique features; while being adaptable for use with all vector systems, they will be particularly well suited to a specific system. To the benefit of scientists, there are a lot of "sports authorities" in the shape of companies that have developed some fantastic technologies for recombinant protein expression. These have been packaged into teams that will help your client make some notable impressions for the fans. You, as an agent, just need to pick up the phone and make those calls. A list of purification systems and companion products for use with the various fusion and tag vectors is found in the accompanying table. Thomas Unger, Vice President of Research and Devlopment for Miragen Inc., a biotech company located in Irvine, CA., can be reached at tfunger@aol.com No Tag Vectors Table One Tagfusion Excision Vectors Table One Tag Vectors Table Two Tagfusion Excision Vectors Table Two Tag Vectors Table Two Tagfusion Excision Vectors Table

Show Me The Money: Prokaryotic Expression Vectors And Purification Systems

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