Prokaryotic Expression Of Human Polypeptide N-Acetylgalactosaminyl Transferase And In Vitro Synthesis Of O-GalNAc Glycosylated Interleukin-2

O-GalNAc glycosylation is an important form of post-translational modification of proteins.It not only participates in protein processing,cell proliferation and differentiation,immune inflammation and other processes,but also has important effects on therapeutic protein drugs.Lack of glycosylation will result in shorter half-life or reduced efficacy of therapeutic protein drugs in vivo.polypeptide N-acetyl-?-galactosaminyltransferases(ppGalNAc-Ts,EC:2.4.1.41)is the initial glycosyltransferase which regulates the O-GalNAc glycosylation of protein.It is also the key glycosyltransferase that regulates O-glycosylation of human proteins and its biological function.Obtaining human-derived ppGalNAc-T in high yield in vitro is of great significance for the synthesis of recombinant therapeutic O-glycoprotein drugs,vaccine production and the development of tool enzymes.Since ppGalNAc-T has only existed in metazoa,there are no isozymes that can substituted it in bacteria and yeast.For a long time,people can only obtain O-GalNAc modified glycopeptides through chemical synthesis of peptides.However,there are still limitations on the synthesis of O-glycan for large molecular weight protein drugs.The use of insect cells or human-derived HEK 293 T cells to express ppGalNAc-T has the problems of low yield and high culture cost,and cannot meet the growing demand for glycoprotein drugs.In the past,when using the prokaryotic system to express human ppGalNAc-T,there were problems such as the need for multiple chaperones to co-express to help the ppGalNAc-T folding,resulting in low expression efficiency.Therefore,in this study,we developed an E.coli expression system that can obtain ppGalNAc-T with a yield of more than 12 mg/L and stable enzyme activity.In this study,Rosetta-gami^TM 2(DE3)pLysS was used as the expression strain.Co-expression of the human protein disulfide isomerase(PDI,EC: 5.3.4.1)can obtain highly active ppGalNAc-T from this E.coli expression strain.The main results are as following:(1)A method for expressing ppGalNAc-T in E.coli with simple operation,high expression and strong enzymatic activity was established.Studies have found that co-expression of recombinant human PDI and ppGalNAc-T2 in the Rosetta-gami^TM 2(DE3)pLysS strain can help the ppGalNAc-T2 form the correct disulfide bond and increase its solubility.After optimizing the expression conditions,more than 12 mg of ppGalNAc-T2 can be obtained from 1 L medium.(2)Using HPLC-based enzyme kinetics experiments,circular dichroism experiments to systematically compare the differences between ppGalNAc-T produced by E.coli and HEK 293 T cell expression systems.We found that E.coli-T2 and 293T-T2 have similar Vmax against EA2 and UDP-GalNAc.But E.coli-T2 has a stronger affinity for EA2 and UDP-GalNAc than 293T-T2.E.coli-T2 has similar thermal stability as293T-T2,but there is a slight difference in the alpha helix structure.(3)In vitro enzymatic synthesis O-GalNAc glycosylated interleukin-2by using E.coli-T2.We further analyzed the O-GalNAc glycosylation site of interleukin-2 by site-directed mutation experiments,and proved the mechanism of ppGalNAc-T's precise regulation of substrate protein glycosylation.In summary,in this study,we constructed a ppGalNAc-T E.coli expression system through co-expression of PDI.This expression system is easy to operate and can produce a large amount of active ppGalNAc-T.Taking IL-2 as an example,we confirmed that the ppGalNAc-T expressed by E.coli can be used to in vitro enzymatic synthesis of O-GalNAc glycan on proteins in vitro.Our study provides technical support for the in vitro enzymatic synthesis of O-glycoprotein drugs in vitro,and similar strategies can be used to try to express other glycosyltransferases.