Study On The Expression And Purification Of GST-p33^ING1b Fusion Protein

Objective: To construct the prokaryotic expression vector of GST-p33^ING1b fusion protein to express the fusion protein in BL21 E.coli strain in high effeciency and establish the purification method under the laborary conditions.Methods: To construct the recombinant plasmid pGEX-5X-3-p33^ING1b, the open reading frame (ORF) of p33^ING1b amplified by PCR was firstly inserted to pBS-T vector and then subcloned to the prokaryotic expression vector pGEX-5X-3. DNA sequencing revealed that the inserted fragment was correct in sequence, orientation and reading frame position. BL21 strain was then tranformed by the recombinant plasmid and expression of the fusion protein was induced by IPTG Finally, the induced strain was ultrasonicated, the inclusion bodies were collected and dissolved by urea, and the target fusion protein was denaturized by urea of high concentration. After renaturized by dialysis, the target protein was released to the supernatant and purified by glutathione sepharose 4B. The immunogenicity of the purified protein was further demonstrated by means of Western blot with anti-GST primary monoclonal antibody.Results: The full length ORF of p33^ING1b was successfully amplified by PCR. The existence of the ORF fragment in the recombinant plasmid pGEX-5X-3-p33^ING1b was demonstrated by restrictive endonuclease digestion. The validity of the fragment in the aspect of sequence, orientation and reading frame position was further demostrated by DNA sequencing. The inserted fragment was 831bps and the theorical molecular weight (MWt) of the fusion protein was about 60kDa. On SDS-PAGE results, a new band consisted with the theorical MWt of GST- p33^ING1b (about 60kDa) could be seen on the lanes of the protein samples of the recombinant plasmid transformed BL21 strain after IPTG induction. The fusion protein could be purificated by glutathione sepharose 4B mediated affmitive chromatography after it was released from the inclusion bodies to the supernatant by urea denaturization and dialysis renaturization. The immumogenictiy of the GST fusion protein was successfully demonstrated by immunoblotting and anti-GST antibody.Conclusions: In this study, we have successfully constructed the prokaryotic expression vector and induced the expression of the fusion protein in BL21 strain. The fusion protein has been purificated efficiently after inclusion body denaturization and renatuarization by affinitive purification.