PGEX vectors are important synthetic genetic construct used for creating engineered GST Tagged proteins in E.Coli in biotech laboratory for research and commercial purposes.
Engineered GST fusion proteins are constructed by inserting a gene or gene fragment into the multiple
cloning site of one of the ten pGEX vectors. This will give genetic engineers having multiple unique choice for their projects. Expression is under the control of the “tac” promoter, which is induced by the lactose analog isopropyl b-D thiogalactoside (IPTG). All pGEX vectors are also engineered with an internal lacI q gene. The lacI q 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 expression of the insert. This give the engineer choices to control expression process based on their needs, condition and standards.
In addition to offering chemically inducible, high-level expression, the vectors allow mild elution conditions for release of fusion proteins from the affinity medium. Thus, effects on antigenicity and functional activity of the protein are minimized. The vectors have a range of protease cleavage recognition sites that lets engineers choose the best suitable vector based on host, protein and purpose. Collectively, the pGEX-P, pGEX-T, and pGEX-X series vectors provide all three translational reading frames beginning with the EcoR I restriction site that give a good opportunity for tagging and inserting sequences. The same multiple cloning sites (MCS) in each vector ensure easy transfer of inserts. pGEX-6P-1, pGEX-4T-1, and pGEX-5X-1 can directly accept and express cDNA inserts isolated from lgt11 libraries. pGEX-2TK has a different MCS from that of the other vectors. pGEX-2TK is uniquely 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 thrombin recognition site and the MCS. Expressed proteins can be directly labelled using protein kinase and [g– 32 P]ATP and readily detected using standard radiometric or autoradiographic techniques.
Based on these information for a listing of the control regions of the pGEX vectors. Complete
DNA sequences and restriction site data are available at the GenBank databases.
pGEX-6P Pre Scission Protease vectors offer the most efficient method for cleavage and
purification of GST fusion proteins in the biotechnology laboratory. Site-specific cleavage is performed with simultaneous immobilization of the protease on the column that make a good resolution in the results. The protease has high activity at low temperature so that all steps can be performed in the cold room to protect the integrity of the target protein for high resolution purification process. Cleavage enzyme and GST tag are removed in a single step that make this technique as a top technique for protein purification in the laboratory.