Study On Prokaryotic Expression Of Human PpGalNAc-T2 And Homology Modelling Of Ricin-like Domain In PpGaINAc-T2

Objective: To express the full-length encoding sequence of human polypeptide N-Acetylgalactosaminyltransferase 2 in Escherichia coli BL21; to simulate the 3D structure of ricin-like domain of ppGalNAc-T2. Methods: The cDNA gene encoding ppGalNAc-T2 was subcloned from plasmid pDONR201-T2 into prokaryotic expression vector pGEX-4T-l, resulting in the recombinant plasmid pGEX-4T-l-T2, which was subsequently transformed into Escherichia coli BL21. IPTG was used to induce the expression of the target gene; Exploit SWISS-MODEL, an automated protein homology-modeling server, to simulate the 3D structure of the ricin-like domain of ppGalNAc-T2 and predict the possible active site through comparing with the temple protein. Results: The plasmid pGEX-4T-l-T2 was reconstructed successfully and a new added Mr 90.7xl03 fusion protein was detected by 12% SDS-PAGE. The fold of the predicted structure is β -trefoil and the second structure is mainly β strand and ASN4, ASP7, ASN28 may constitute the sugar-binding site, which may play an essential role in the enzyme activity of ppGalNAc-T2. Conclusion: We have successfully expressed the ppGalNAc-T2 gene in Escherichia coli BL21, which establishes a foundation for the further research in detecting the enzyme activity of ppGalNAc-T2; We have obtained thepredicted 3D structure of the ricin-like domain of ppGalNAc-T2, which provides evidence for the role played by such domain in the enzyme activity of ppGalNAc-T2.