PGEX-4T-1
A tac promoter for chemically inducible, highlevel expression of GSTtagged recombinant proteins.
An internal lacIq gene for use in any E. coli host.
Very mild elution conditions for release of fusion proteins from the affinity matrix, thus minimizing effects on antigenicity and functional activity.
PreScission Protease, Thrombin, or Factor Xa recognition sites for cleaving the desired protein from the fusion product.
Thirteen pGEX vectors are available (see Figure). 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. pGEX6P1, pGEX6P2, and pGEX6P3 each encode the recognition sequence for sitespecific cleavage by PreScission Protease, (see PreScission Protease) between the GST domain and the multiple cloning site. pGEX4T1, pGEX4T2, and pGEX4T3 are derived from pGEX2T and contain a Thrombin recognition site. pGEX5X1, pGEX5X2, and pGEX5X3 are derivatives of pGEX3X and possess a Factor Xa recognition site.
pGEX2TK is designed to allow the detection of expressed proteins by directly labeling the fusion products in vitro (1). This vector contains the recognition sequence for the catalytic subunit of cAMPdependent 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 [gamma32P]ATP and readily detected using standard radiometric or autoradiographic techniques. pGEX2TK is a derivative of pGEX2T; its fusion proteins can be cleaved with Thrombin.
Cleavage of pGEX6P GST fusion proteins occurs between the Gln and Gly residues of the recognition sequence LeuGluValLeuPheGlnGlyPro (2). Low temperature (5°C) digestion minimizes the degradation of the protein of interest. Because PreScission Protease has been engineered with a GST tag, it can also be removed from the cleavage mixture simultaneously with the GST portion of the fusion protein. The pGEX6P Expression Vectors permit convenient sitespecific cleavage and simultaneous purification on Glutathione Sepharose. The pGEX6P series provides all three translational reading frames linked between the GST coding region and the multiple cloning site. Collectively, the pGEX vectors provide all three translational reading frames beginning with the EcoRI restriction site. pGEX1?T, pGEX6P1, pGEX4T1, and pGEX5X1 can directly accept and express cDNA inserts isolated from ?gt11 libraries.References1. Kaelin, W.G. et al. Cell 7NA, 351 (1992).