Gene Expression

GST Gene Fusion System
Glutathione Sepharose 4B
HisTrap Kit
Recombinant Phage Antibody System (RPAS) Overview
Mouse ScFv Module
Expression Module
Detection Module
Recombinant Phage Selection Module
RPAS Purification Module
U.S.E. Mutagenesis Kit

GST Gene Fusion System

• For high-level expression of cloned genes and purification of functional proteins.
• Features the enzyme glutathione S-transferase from Schistosoma japonicum as the affinity handle for purifying fusion proteins.
• Uses the inducible tac promoter to produce high levels of gene expression in E. coli.
• Utilizes immobilized glutathione for affinity purification of expression products.
• Provides mild elution conditions during purification to maintain protein antigenicity and functional activity.
• Features the protease cleavage sites for thrombin, factor Xa and PreScission Protease to allow separation of the protein of interest from the GST fusion partner.
• Provides a colorimetric assay and an immunoassay for detecting expressed fusion proteins.

pGEX Vectors

Site-specific Proteases

PreScission Protease

Thrombin

Factor Xa

Host Strains

E. coli BL21

Detection Reagents

Anti-GST Antibody

The Glutathione S-transferase (GST) Gene Fusion System provides a complete system for expressing and purifying fusion proteins in E. coli. The System utilizes plasmid vectors designed for inducible, high-level expression of genes or gene fragments as fusion proteins with the 26-kDa GST domain from S. japonicum (1).

Expressed fusion proteins are easily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Cleavage of the desired protein from the fusion product is achieved using a site-specific protease whose recognition sequence is located immediately upstream from the multiple cloning site (MCS). The GST System has been used successfully in many applications such as molecular immunology (2), the production of vaccines (3, 4) and studies involving protein-protein (5) and DNA-protein interactions (6).

pGEX Vectors
The GST System consists of thirteen expression vectors, two Purification Modules, and a Detection Module. The vectors collectively cover all three translational reading frames. pGEX-2TK contains the recognition site for the catalytic subunit of cAMP-dependent protein kinase. This site allows direct labelling of fusion proteins using [gamma-³²P]ATP for autoradiographic detection (7).

Purification Modules

Fig. 1. Diagram showing overview of the GST Purification Modules.

Two GST Purification Modules feature Glutathione Sepharose 4B packaged in different formats. The RediPack GST Purification Module provides convenient disposable columns pre-packed with 2 ml of Glutathione Sepharose 4B, sufficient for two purifications using 2 ml samples.

The Bulk GST Purification Module provides a 10 ml bulk pack of Glutathione Sepharose 4B and five disposable columns. With this module, fusion proteins can be purified using either column chromatography or a batch method. The batch method is more flexible, as purification can be performed with 100 µl (7) to 2 ml (1) of Glutathione Sepharose 4B. The Bulk Module contains enough reagents for five purifications using 2 ml samples.

Each module also contains IPTG for induction of expression, reduced glutathione for elution of fusion proteins from the Glutathione Sepharose 4B, thrombin for cleavage of fusion proteins containing the thrombin recognition site, and a detailed instruction booklet.

Detection Module

Fig. 2. Diagram of the enzymatic assay for glutathione S-transferase.

The GST Detection Module is designed to identify GST fusion proteins using either a biochemical or immunological assay. The biochemical assay utilizes glutathione and 1-chloro-2-4-dinitrobenzene (CDNB) as substrates for GST. This reaction yields a yellow product detectable at 340 nm (8). The immunological assay uses Anti-GST Antibody suitable for use with Western blots. The GST Detection Module contains enough reagents for 50 detection reactions using either assay and includes:

1-chloro-2-4-dinitrobenzene
Glutathione
Anti-GST Antibody
Instruction booklet

Fig. 3. Diagram of the immunological assay used to detect glutathione S-transferase fusion proteins.

Anti-GST Antibody

• For the highly sensitive and specific detection of recombinant schistosomal GST fusion proteins expressed using pGEX vectors.
• Extensively cross-adsorbed to remove antibodies that bind to E. coli proteins and affinity-purified using GST-Sepharose.
• Supplied unconjugated for use with any enzyme-conjugated anti-goat antibody.
• Recommended for use in Western blots and dot blots to detect GST fusion proteins.

Product	Quantity	Code No.
Anti-GST Antibody (50 detections)	0.5 ml	27-4577-01
Companion Products
GST Detection Module		27-4590-01

REFERENCES

1. Smith, D. B. and Johnson, K. S.,Gene 67, 31 (1988).
2. Toye, B.et al., Infect. Immun. 58, 3909 (1990).
3. Fikrig, E.et al., Science 250, 553 (1990).
4. Johnson, K. S. et al., Nature 338, 585 (1989).
5. Kaelin, W. G. et al., Cell 64, 521 (1991).
6. Kaelin, W. G. et al., Cell 65, 1073 (1991).
7. Kaelin, W. G. et al., Cell 70, 351 (1992).
8. Habig, W. H. et al., J. Biol. Chem. 249, 7130 (1974).

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Glutathione Sepharose 4B

• For the purification of recombinant fusion proteins containing the carboxyl terminus of glutathione S-transferase Schistosoma japonicum as an affinity tail.
  
• Also ideal for purification of other S-transferases or glutathione-dependent proteins.
  
• The 100 ml pack size is function-tested (see below).

Glutathione Sepharose 4B consists of the ligand glutathione, coupled via a 10-carbon spacer arm to the oxirane group of epoxy-activated Sepharose 4B.

Glutathione Sepharose 4B is available in two forms:
Bulk media: for the purification of larger quantities.
Pre-packed disposable columns: for gravity-flow affinity chromatography. Each pre-packed column contains 2 ml of the gel and is supplied with detailed instructions.
The 100 ml pack size is function-tested for binding of recombinant GST. Minimally, each ml of drained gel binds 8 mg of recombinant GST.

The gel combines the high chemical stability of the well-known Sepharose 4B matrix (wet bead diameter ~ 45-165 µm) with a binding capacity of > 5.0 mg horse liver glutathione S-transferase per ml of drained gel.

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Recombinant Phage Antibody System

• For cloning and expression of antibody gene variable domains from mice, using a novel expression system allowing both phage-display and soluble expression.
• Fast, inexpensive production of recombinant antibodies for use in immunological applications.
• Immortalizes antibody genes as a stable source of genetic material.
• Provides the technology for constructing novel antibodies or for modifying antibody affinity and specificity.

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Technical Information

The Recombinant Phage Antibody System (RPAS) is an integrated modular system designed for the cloning and expression of recombinant antibody fragments in bacteria. The technology, developed in collaboration with Cambridge Antibody Technology Ltd., UK, relies on a phage-display system in which fragments of antibodies are expressed as fusion proteins displayed on the phage surface. With this system, it becomes technically feasible to produce antibodies quickly (under optimal conditions this can be accomplished in eight days) in bacterial cultures and to genetically manipulate their structure. RPAS allows the expression of soluble recombinant antibodies for use as immunological reagents, as well as the production of phage-displayed recombinant antibodies for affinity enrichment against the antigen of interest. The core system is comprised of three integrated modules:

• Mouse ScFv Module
• Expression Module
• Detection Module

Optional modules which may be used in conjunction with the core system include:

• Recombinant Phage Selection Module
• RPAS Purification Module

A wide range of support products are also available for use with RPAS.

• HRP/Anti-E Tag Conjugate

• Anti-E Tag Antibody

• Light Primer Mix
• Heavy Primers
• Linker Primer Mix
• RS Primer Mix

As an added convenience to purchasers of the Mouse ScFv Module, the four primer sets included in the kit are also available as stand-alone products. The primers are provided at concentrations identical to those found in the module to allow for direct substitution.

• Anti-M13 Antibody
• HRP/Anti-M13 Conjugate

Anti-M13 Antibody is designed for use in immunoassays in an unconjugated form or for conjugation to a reporter molecule (e.g. alkaline phosphatase). HRP/Anti-M13 Conjugate is available for high volume users. It is provided at a concentration identical to that found in the Detection Module.

• pCANTAB 5 Gene Rescue Primers
• pCANTAB 5 Sequencing Primer Set

These primers are designed to aid in the analysis and genetic manipulation of the ScFv fragments produced using the system. The Gene Rescue Primers are used in PCR to amplify the entire ScFv insert from the vector. The Sequencing Primer Set contains two sets of primers designed for sequencing the VH and VL regions of the ScFv fragment.

RELATED REFERENCES

1. Marks, J. D. et al., Biotechnology 10, 779 (1992).
2. Winter, G. and Milstein, C., Nature 349, 293 (1991).
3. Clackson, T. et al., Nature 352, 624 (1991).
4. Marks, J. D. et al., J. Mol. Biol. 222, 581 (1991).
5. Chaudhary, V. K. et al., Proc. Natl. Acad. Sci. USA 87, 1066 (1990).
6. Chiswell, D. J. et al. TIBTECH 10, 80 (1992).
7. McCafferty, J. et al., Nature 348, 552 (1990).
8. Huston, J. S. et al., Proc. Natl. Acad. Sci.USA 85, 5879 (1988).

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TECHNICAL INFORMATION

Fig. 4. Schematic overview of the Recombinant Phage Antibody System

Mouse ScFv Module

The Mouse ScFv Module is designed to produce a repertoire of single-chain variable fragment (ScFv) genes in which the variable regions of the antibody heavy (VH) and light (VL) chain genes are joined by a flexible linker.

Using mRNA isolated from mouse hybridoma or spleen cells, the Primed First-Strand Reaction Mix generates first-strand cDNA through random hexamer priming of the mRNA template. Since the specific antibody VH and VL genes represent only a small fraction of the total cDNA, they must first be amplified to generate sufficient DNA for cloning.

The heavy and light chain genes are amplified in two separate PCR** reactions using primers* specific for the variable region of each chain. After amplification, the PCR products are purified separately by agarose gel electrophoresis to remove primers and unwanted products.

The purified heavy and light chain DNA products are then assembled into a single gene using a DNA linker fragment, constructed such that its ends anneal to the 3' end of the heavy chain and the 5' end of the light chain PCR products. The assembly reaction ultimately produces a small amount of the ScFv gene (750 bp) where the VH region is linked to the VL region through a sequence encoding a (Gly₄Ser) ₃ peptide. This arrangement maintains the correct reading frame.

The assembled ScFv DNA fragment is then amplified using a set of restriction site primers that add Sfi I and Not I sites respectively to its 5' and 3' ends. Since these sites normally occur in antibody genes at a very low frequency, most ScFv genes can be cloned as a single Sfi I/Not I fragment. Following amplification, the ScFv DNA fragment is purified on a Sephacryl S-400 HR MicroSpin Column and sequentially digested with Sfi I and Not I to generate cohesive ends for ligation into the cloning vector. After a second column purification to remove small Sfi I/Not I fragments, the ScFv DNA fragment can be ligated into pCANTAB 5 E using the Expression Module.

Mouse ScFv Module contains sufficient reagents for five amplifications:

Primed First-Strand	Linker-Primer Mix
Reaction Mixes	RS Primer Mix
Control mRNA	Sephacryl S-400 HR
Heavy Primer 1	ScFv Marker
Heavy Primer 2	MicroSpin Columns
VH Marker	DTT Solution
Light Primer Mix	RNase-Free Water

**The PCR process is covered by U.S. patents 4,683,195 and 4,683,202 owned by Hoffman-La Roche Inc. Use of the PCR process requires a license. Nothing in this literature should be construed as an authorization or implicit license to practice PCR under any patents owned by Hoffman-LaRoche.
*Lambda light chain primers are not included in the Mouse ScFv Module for two reasons: their sequences are not well characterized, and these chains are expressed infrequently in mice. Consequently, lambda light chains may not be amplified.
MicroSpin is a trademark of Lida Manufacturing Corp.
Aspects of this technology are licensed and/or patented.

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Expression Module

The Expression Module is designed to clone the antibody ScFv gene, synthesized in the Mouse ScFv Module, into a phagemid and to express phage-displayed recombinant antibodies.

The pCANTAB 5 E phagemid contains restriction-digested Sfi I and Not I sites such that the ScFv gene can be cloned in defined orientation. After ligation to the ScFv insert, the phagmid is used to transform competent E. coli TG1 cells. The transformed cells are then infected with M13KO7 helper phage to rescue the phagemid and antibody ScFv gene. The resulting recombinant phage contain a single-strand copy of the phagemid DNA encoding a specific antibody ScFv gene. They will display one or more copies of the corresponding recombinant antibody as fusion proteins on their tips.

These displayed antibody fragments are fully functional and will bind antigen. This feature allows for selection against a target antigen in an affinity purification that enriches for those phage bearing ScFv capable of reacting with the target. Selection can be performed either on a solid phase as described in the instructions which accompany the Expression Module or in solution using biotinylated antigen according to the protocol provided with the Recombinant Phage Selection Module. Since the ScFv is attached to the phage particle, the entire phage is immobilized to the antigen; therefore, both the ScFv and the genes coding for its expression are purified from non-reactive phage. This feature is especially useful for studying the relationship of gene structure and antibody function, as the M13 system is particularly amenable to DNA sequencing and mutagenesis.

Once an antigen-positive clone has been identified and isolated, recombinant phage may be used to infect E. coli HB2151 cells, which will recognize an amber translational stop codon encoded by the pCANTAB 5 E vector. Soluble antibodies can be produced in this host strain for use as immunological reagents. If desired, soluble ScFvs may be selectively purified from other E. coli proteins using the RPAS Purification Module.

Expression Module contains:

pCANTAB 5 E Expression Vector	ScFv Marker
Control Insert	10X One-Phor All Buffer PLUS
M13KO7 Helper Phage	E. coli TG1 Cells
E. coli HB2151 Cells

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TECHNICAL INFORMATION

Control regions and restriction site map for pCANTAB 5 E.

Fig. 5. Schematic diagram pCANTAB 5 E phagemid.

Detection Module

The Detection Module utilizes an enzyme-linked immunosorbent assay (ELISA) to detect and identify phage-displayed recombinant antibodies. Antigen is coated onto the surface of wells in a microtiter plate. Any remaining sites on the plastic surface are blocked with excess protein to prevent non-specific adsorption of phage antibodies to the plastic. Phage antibody is then added and allowed to interact with the antigen. The plate is washed, and the HRP/Anti-M13 Conjugate, which binds to the phage coat protein, is added to detect the presence of antigen-positive recombinant phage. After washing to remove unbound conjugate, the ABTS substrate is added. A positive reaction is indicated by the development of a green color.
Detection Module contains sufficient reagents for performing ELISAs on twenty 96-well microtiter plates:

HRP/Anti-M13 Conjugate

Positive Control Phage

Positive Control Antigen

ABTS

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Recombinant Phage Selection Module

The Recombinant Phage Selection Module is designed to provide an alternative method for selecting recombinant phage antibodies using biotinylated antigen in a solution-phase system. The amount of antigen used in solution-based selection is easy to control; therefore, based on the quantity of antigen used, one can select for phage-displayed antibodies which exhibit desired affinities or kinetics of dissociation.

Using the Recombinant Phage Selection Module, protein antigens which contain a free amino group are biotinylated with NHS-LC-biotin. After removing unincorporated biotin and determining protein recovery and percent biotinylation, the biotinylated antigens are incubated with recombinant phage-displayed antibodies in solution. Knowing the amount of protein and its degree of biotinylation allows for more accurate control of the antigen concentration. By limiting the amount of antigen used for selection, one can therefore progressively select for antibodies with higher affinities (1, 2, 3).

Once formed, the biotinylated antigen/recombinant phage antibody complex is captured from solution using streptavidin coupled to a crosslinked beaded agarose. By washing the agarose, non-reactive phage antibodies are removed, leaving only those phage antibodies which specifically recognize the antigen. This selection process enriches for antigen-reactive phage antibodies.

Recombinant Phage Selection Module contains:

Biotin Reagent	BSA
Biotinylated Control	Streptavidin Agarose
Sephadex G-25 DNA Grade	MicroSpin Columns
PBS	PEG Buffer
Carbonate Buffer	HABA Reagent
Avidin	Pronase

Pronase is a trademark of Calbiochem.

REFERENCES

1. Parmley, S. P. and Smith, G. P., Gene 73, 305 (1988).
2. Bass, S., Greene, R. and Wells, J. A., Proteins: Structure, Function, and Genetics 8, 309 (1990).
3. Hawkins, R. E., Russell, S. J. and Winter, G., J. Mol. Biol. 226, 889 (1992).

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TECHNICAL INFORMATION

Fig. 6. Schematic showing protocol of the Recombinant Phage Selection Module.

RPAS Purification Module

The RPAS Purification Module is a kit designed for rapid, efficient affinity purification of "E-tagged" soluble ScFv recombinant antibodies produced in E. coli. The Module provides all of the reagents necessary to perform a complete one-step purification without using complicated equipment. It contains a 5 ml HiTrap column pre-packed with Anti-E Tag Sepharose High Performance medium and pre-formulated Binding, Elution and Neutralizing buffers, as well as a syringe and connectors. Separations are easily performed using the supplied syringe or, alternatively, with a laboratory pump. Complete easy-to-follow instructions for use and sample preparation are included.

When ScFv inserts are cloned into the pCANTAB 5 E vector, functional soluble ScFv-fragments bearing a peptide tag ("E-Tag") can be expressed in the E. coli periplasm or may be secreted into the culture medium (see Expression Module). E-tagged soluble ScFvs from the sample selectively bind to the Anti-E Tag column at neutral pH. The column is washed to remove nonspecifically bound proteins and ScFv are easily eluted by lowering the pH. Fractions are collected and can be assayed for activity following neutralization.

RPAS Purification Module contains:

HiTrap Anti-E Tag Column
Binding Buffer
Elution Buffer
Neutralizing Buffer
Connectors
Luerlock female/M6 male
Luerlock female/M6 female
Tubing connector flangeless/M6 male
Tubing connector flangeless/M6 female
Syringe
Domed nut
Instructions

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Unique Site Elimination (U.S.E.) Mutagenesis

• For site-directed mutagenesis of cloned genes using a plasmid of choice.
• Can be used with any double-stranded plasmid that contains unique, non-essential restriction sites.
• Achieves mutation frequencies of up to 80% or greater using the control reaction.
• Utilizes simple restriction enzyme digestion as the basis for mutant selection.
• Can be used with separate selection/toggle primer sets, thus making the kit compatible with most available plasmids.
• Does not require subcloning of DNA inserts or special host-vector systems when incorporating successive mutations.

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Technical Information

U.S.E. Mutagenesis Kit is designed for rapid site-specific mutagenesis of virtually any double-stranded plasmid. The kit is based on the unique site elimination (U.S.E.) method developed by Deng and Nickoloff at Harvard University (1). The method utilizes a target mutagenic primer and a selection primer to introduce site-specific mutations into the plasmid. Both mutations are subsequently incorporated into the same strand by in vitro DNA synthesis, using T4 DNA polymerase.

Fig. 7. Diagram of the U.S.E. mutagenesis procedure.

The target mutagenic primer is complementary to the gene of interest and carries the desired mutation. The selection primer is directed to a unique restriction site in a non-essential region of the recombinant plasmid. The mutation which this primer introduces will alter the enzyme recognition sequence and, in effect, eliminate it - hence, the name "unique site elimination". During a brief annealing step, both primers hybridize to one strand of the denatured plasmid DNA, thus priming it for DNA synthesis. In vitro DNA synthesis is accomplished using the convenient pre-formulated Reaction Mix and Nucleotide Mix which provide FPLCpure T4 DNA Polymerase, FPLCpure T4 DNA Ligase, gene 32 protein and nucleotides. The newly synthesized DNA is then treated with the restriction enzyme whose site was mutated by incorporating the U.S.E. selection primer. Plasmid DNA that contains the U.S.E. selection primer will no longer have the proper recognition sequence for the restriction enzyme and will be resistant to digestion. Plasmid DNA that does not possess the U.S.E. selection primer will be linearized.

The newly synthesized and mutagenized double-stranded DNA is then introduced by transformation into E. coli strain NM522 mutS, which is defective in repairing mismatches in duplexed DNA. This defect helps to ensure that the mutations introduced by the primers during in vitro synthesis remain stable during transformation. Since covalently closed circular DNA will be taken up by bacteria 100- to 1000-fold more efficiently than linear DNA (2), the majority of the transformants will contain mutant plasmids.

A plasmid preparation from this culture can then undergo an additional round of restriction enzyme selection to increase the proportion of mutant plasmids. After the second round of selection, an appropriate E. coli host is transformed with the restricted plasmid preparation.

Two sets of U.S.E. Selection/Toggle Primers are available separately which can be used on most pUC- or pBR-derived plasmids. One primer converts the Sca I site in the ampicillin-resistance gene to an Mlu I site. Another selection primer modifies the Ssp I site into a Stu I site. Neither mutation affects ampicillin resistance or plasmid function. Corresponding toggle primers can be used to reinstate the original sites and enable successive rounds of mutagenesis.

The pGEX U.S.E. Primers allow the rapid mutagenesis of DNA inserts that are cloned into any of the pGEX series of bacterial expression vectors. When used with U.S.E. Mutagenesis Kit, the primer sets enable a series of site-specific mutations to be successively incorporated into cloned gene sequences without subcloning. Mutated genes can then be expressed and the altered proteins purified under non-denaturing conditions for structure/function studies using the GST Expression System. The frequency of selecting plasmids containing both primer-directed mutations depends on several factors including:

• Quality of the double-stranded plasmid DNA, and the target mutagenic and selection primers.
• Quality of the restriction enzymes used during the selection process.
• Competency of the cells used during the initial transformation.

Mutation frequencies of up to 80% or greater are routinely achieved using the control reaction provided with the kit.

Each kit contains enough reagents for 25 individual mutagenesis reactions and four control reactions. The components include:

One-Phor-All Buffer PLUS

Nucleotide Mix

Reaction Mix

Control Plasmid

Control Mutagenic Primer

Control Selection Primer

E. coli NM522 mutS

Instruction booklet

REFERENCES

1. Deng, W. P. and Nickoloff, J. A., Anal. Biochem. 200, 81 (1992).
2. Conley, E. C. and Saunders, J. R., Mol. Gen. Genet. 194, 211 (1984).

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TECHNICAL INFORMATION