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  • imagination at work

    GE Healthcare

    Data file 28-9622-84 AA GST Gene Fusion System

    Glutathione S-transferase (GST) Gene Fusion SystemThe Glutathione S-transferase (GST) Gene Fusion System from GE Healthcare is a versatile system for the expression, purification, and detection of GST-tagged proteins produced in E. coli. The system consists of three major components: pGEX plasmid expression vectors, products for GST purification, and a variety of GST detection products. A series of site-specific proteases for cleavage of the GST tag complements the system. The GST affinity tag permits a mild purification process that does not affect a proteins native structure and function.

    GST Gene Fusion System benefits include:

    All pGEX vectors offer a tac promoter for chemically inducible, high-level expression

    Mild, nondenaturing buffer compositions for isolation of active proteins

    Affinity chromatography products based on Glutathione Sepharose media for one-step purification of samples from low microgram to gram scale

    Convenient prepacked formats suitable for single samples or parallel screening of multiple cloning constructs

    PreScission Protease, Thrombin, or Factor Xa recognition sites on pGEX vectors for cleaving the target protein from the fusion product

    Easy detection of fusion protein using Anti-GST Antibody

    GST occurs naturally as an Mr 26 000 protein, which can be expressed in E. coli with full enzymatic activity. The crystal structure of recombinant Schistosoma japonicum GST from pGEX vectors has been determined and matches that of the native protein. The pGEX plasmid vectors are designed for inducible, high-level intracellular expression of genes or gene fragments as fusions with S. japonicum GST.

    Recombinant proteins are easily purified from, for example E. coli cell lysates by affinity chromatography using Glutathione Sepharose media (Fig 1), either with prepacked columns or by batch purification.

    Cleavage of the target protein from GST is achieved using a site-specific protease, which possesses a recognition sequence located immediately upstream from the multiple cloning site on the pGEX vectors. Cleavage of the GST tag is performed on-column as a part of the purification protocol or off-line after purification. Recombinant proteins can be detected using an immunoassay provided in the GST Detection Module, Western blotting with Anti-GST Antibody, or by a colorimetric assay.

    pGEX vectorsGST-tagged proteins are constructed by inserting a gene or gene fragment into the multiple cloning site of one of the pGEX vectors. The vectors provide all three translational reading frames beginning with the EcoRI restriction site (see Table 1).

    Fig 1. Glutathione Sepharose High Performance, Glutathione Sepharose 4 Fast Flow, and Glutahione Sepharose 4B are all available as bulk media and in prepacked columns and multiwell plates for purification of GST-tagged proteins.

  • 2 28-9622-84 AA

    Table 1. Protease cleavage sites of pGEX vectors

    Vector Cleavage enzymepGEX-6P-1, pGEX-6P-2, pGEX-6P-3 PreScission ProteasepGEX-4T-1, pGEX-4T-2, pGEX-4T-3 ThrombinpGEX-5X-1, pGEX-5X-2, pGEX-5X-3 Factor XapGEX-2TK Thrombin

    Nine of the vectors have an expanded multiple cloning site (MCS) that contains six restriction sites. The expanded MCS facilitates the unidirectional cloning of cDNA inserts obtained from libraries constructed using many available lambda vectors. pGEX-6P-1, pGEX-6P-2, and pGEX-6P-3 each encode the recognition sequence for site-specific cleavage by PreScission Protease between the GST domain and the multiple cloning site. pGEX-4T-1, pGEX-4T-2, and pGEX-4T-3 are derived from pGEX-2TK and contain a Thrombin recognition site. pGEX-5X-1, pGEX-5X-2, and pGEX-5X-3 are derivatives of pGEX-3X and possess a Factor Xa recognition site (see Table 1).

    pGEX-2TK is uniquely designed to allow the detection of expressed proteins by directly labeling the fusion products in vitro. This vector contains the recognition sequence for the catalytic subunit of cAMP-dependent protein kinase obtained from heart muscle. The protein kinase site is located between the GST domain and the MCS. Expressed proteins can be directly labeled using protein kinase and [-32P]ATP and readily detected using standard radiometric or autoradiographic techniques. pGEX-2TK is a derivative of pGEX-2T; its fusion proteins can be cleaved with Thrombin. Collectively, the pGEX vectors provide all three translational reading frames beginning with the EcoRI restriction site. pGEX-1T, pGEX-6P-1, pGEX-4T-1, and pGEX-5X-1 can directly accept and express cDNA inserts isolated from gt11 libraries.

    To complement the pGEX vectors, GST Vector Primers for Sequencing are available for immediate use in sequencing double-stranded DNA inserted into the pGEX vectors.

    A wide variety of E. coli host strains can be used for cloning and expression with the pGEX vectors. A lyophilized protease-deficient E. coli host strain for optimal expression of recombinant protein, E. coli BL21, is available separately.

    GST-tagged protein purification and screeningGST-tagged proteins are easily purified from, for example bacterial lysates by affinity chromatography using glutathione immobilized to a matrix. The high specificity between GST and glutathione ensures that high purity is obtained in a single step. Elution is performed under mild, nondenaturing conditions so that protein antigenicity and function is preserved. A variety of affinity chromatography products are available from GE Healthcare. Glutathione Sepharose media (Table 2) are available in several formats ranging from prefilled MultiTrap 96-well filter plates to prepacked, SpinTrap, GraviTrap, HiTrap, and HiPrep

    pGEX-1T

    pGEX-6P-1

    EcoRI SmaI SalI XhoI NotIBamHI

    PreScission Protease

    Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro HisCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT

    pGEX-6P-2

    BamHI

    PreScission Protease

    Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG

    EcoRI SmaI SalI XhoI NotI

    pGEX-6P-3

    BamHI

    PreScission Protease

    Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly ArgCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC

    EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    EcoRI

    CTG GTT CCG CGT GGA TCC CCG GAA TTC ATC GTG ACT GAC TGA CGA

    BamHI

    Leu Val Pro Arg Gly Ser Pro Glu Phe Ile Val Thr Asp

    Thrombin

    Stop codons

    pGEX~4900 bp

    pBR322ori

    BalI

    BspMI Ptac

    cal I q

    NarI

    EcoRV

    BssHII

    BstEII MluI

    ApaI

    Tth111I AatII

    PstI

    p4.5AlwNI

    pSj10 Bam7Stop7

    pGEX-4T-2

    pGEX-5X-1

    pGEX-5X-2

    pGEX-5X-3

    pGEX-4T-1

    pGEX-4T-3

    pGEX-3X

    pGEX-2TK

    Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerCTG GTT CCG CGT GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA

    Stop codon

    Ile Glu Gly Arg Gly Ile Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg AspATC GAA GGT CGT GGG ATC CCC GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA

    Stop codons

    Ile Glu Gly Arg Gly Ile Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerATC GAA GGT CGT GGG ATC CCC GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA

    Stop codon

    Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr AspATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA

    Stop codons

    Leu Val Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg AspCTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA

    Stop codons

    Leu Val Pro Arg Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr AspCTG GTT CCG CGT GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA

    Stop codons

    ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGA CTG ACT GACIle Glu Gly Arg Gly Ile Pro Gly Asn Ser Ser

    Stop codons

    Leu Val Pro Arg Gly Ser Arg Arg Ala Ser Val

    Kinase

    CTG GTT CCG CGT GGA TCT CGT CGT GCA TCT GTT GGA TCC CCG GGA ATT CAT CGT GAC TGA

    Stop codons

    Thrombin

    Thrombin

    Thrombin

    Thrombin

    Factor Xa

    Factor Xa

    Factor Xa

    Factor Xa

    EcoRIBamHI SmaI

    EcoRIBamHI SmaI

    pGEX-2T

    CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACGLeu Val Pro Arg Gly Ser Pro Gly Ile His Arg Asp

    Stop codonsEcoRI

    Thrombin

    BamHI SmaI

    glutat

    hione

    S-transf

    erase

    Amp r

    Fig 2. Map of the glutathione S-transferase fusion vectors showing the reading frames and main features. All thirteen vectors have stop codons in all three frames downstream from the multiple cloning site (not depicted in this map).

    The pGEX vectors are designed for inducible, high-level intracellular expression of genes or gene fragments. Expression in E. coli yields tagged proteins with the GST moiety at the amino terminus and the protein of interest at the carboxyl terminus. Thirteen pGEX vectors are available (see Fig 2); all of them have a tac promot

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