The pGEX 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-2T and contain a Thrombin recognition site. pGEX-5X-1, pGEX-5X-2, and pGEX5X-3 are derivatives of pGEX-3X and possess a Factor Xa recognition site.
- A tac promoter for chemically inducible, high-level expression of GST-tagged 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 minimising effects on antigenicity and functional activity
- PreScission Protease, Thrombin, or Factor Xa recognition sites for cleaving the desired protein from the fusion product
pGEX-2TK is designed to allow the detection of expressed proteins by directly labelling 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 labelled using protein kinase and [gamma-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.
Cleavage of pGEX-6P GST fusion proteins occurs between the Gln and Gly residues of the recognition sequence Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro. Low temperature (5 °C) digestion minimises 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 pGEX-6P Expression Vectors permit convenient site-specific cleavage and simultaneous purification on Glutathione Sepharose. The pGEX-6P 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. pGEX-1λT, pGEX-6P-1, pGEX-4T-1, and pGEX-5X-1 can directly accept and express cDNA inserts isolated from λgt11 libraries.
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 (see 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-2T and contain a Thrombin recognition site. pGEX-5X-1, pGEX-5X-2 and pGEX5X-3 are derivatives of pGEX-3X and possess a Factor Xa recognition site.