GST-tagged proteins are constructed by inserting a gene or gene fragment into the MCS of one of the 13 pGEX vectors. Expression is under the control of the tac promoter, which is induced by the lactose analog isopropyl β-D-thiogalactoside (IPTG). All pGEX vectors are also engineered with an internal lacIq gene. The lace gene product is a repressor protein that binds to the operator region of the tac promoter, preventing expression until induction by IPTG, thus maintaining tight control over the expression of the insert.
Nine of the vectors have an expanded 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 MCS. 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 pGEX-5X-3 are derivatives of pGEX-3X and possess a Factor Xa recognition site.
pGEX-2TK has a different MCS from that of the other vectors. pGEX-2TK is designed to allow the detection of expressed proteins by directly labeling the tagged products in vitro. This vector contains the recognition sequence for the catalytic subunit of cAMP-dependent protein kinase obtained from the heart muscle. The protein kinase site is located between the thrombin recognition site and the MCS. Expressed proteins can be directly labeled using protein kinase and [γ–32P]ATP and readily detected using standard radiometric or autoradiographic techniques. pGEX-2TK is a derivative of pGEX-2T, and its tagged protein can be cleaved with thrombin.
Collectively, the pGEX vectors provide all three translational reading frames beginning with the EcoRI restriction site (Figure 1.1). pGEX-1λT, pGEX-6P-1, pGEX-4T-1, and pGEX-5X-1 can directly accept and express cDNA inserts isolated from λgt11 libraries.