Prokaryotic Expression,Purification and Activity Assay of Human Polypeptide: N-acetylgalactosaminyltransferase2

Objective: To express the full-length encoding sequence of human polypeptide: N-Acetylgalactosaminyltransferase2 in Escherichia coli using vector pGEX-5X-3,and analyze its enzyme activity after purification and renaturation.Methods: The cDNA encoding ppGalNAc-T2,which was amplified from plasmid pDONR201-T2 by PCR,was subcloned into prokaryotic expression vector pGEX-5X-3,resulting in the recombinant plasmid pGEX-5X-3/T2 which was subsequently transformed into Escherichia coli BL21.A fusion protein was expressed after the induction by IPTG.After testified by western blot,we purified the expression products by Glutathione-Sepharose affinity chromatography.After renaturation,its enzyme activity was assayed by Reverse Phase High Performance Liquid Chromatography(RP-HPLC).Results: The plasmid pGEX-5X-3/T2 was reconstructed successfully.The fusion protein was expressed and testified by SDS-PAGE and western blot.The purified enzyme was obtained by affinity chromatography and the HPLC showed its activity.Conclusion: The ppGalNAc-T2 which has certain enzyme activity was successfully expressed,purified and renaturized.