文献类型：期刊文献

英文题名：A study on enhanced O-glycosylation strategy for improved production of recombinant human chorionic gonadotropin in Chinese hamster ovary cells

作者：Deng, Zhe[1];Yi, Xiaoping[1];Chu, Ju[1];Zhuang, Yingping[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200137, Peoples R China

年份：2019

卷号：306

起止页码：159

外文期刊名：JOURNAL OF BIOTECHNOLOGY

收录：;EI(收录号：20194307565242);WOS:【SCI-EXPANDED(收录号:WOS:000496193800020)】；

基金：This work was supported by grants from the Chinese National Programs for High Technology Research and Development (2015AA020801)

语种：英文

外文关键词：Recombinant human chorionic gonadotropin; CHO cell; O-glycosylation; GalNAc; Ugp2; Galnt1

摘要：Human chorionic gonadotropin (hCG) is a glycoprotein hormone that exists as a heterodimer comprised of an a subunit and beta subunit linked with disulfide bridges. The beta subunit contains four O-glycosylation sites. Previous studies have found that the translation of mRNA to polypeptides of the beta subunit was a severely limiting step for the expression of recombinant hCG protein in Chinese hamster ovary (CHO) cells. The effects of O-glycosylation on recombinant hCG protein expression were assessed by adding O-glycan precursors and overexpressing and knocking down key regulatory genes of O-glycan precursor synthesis and O-glycan sugar chain synthesis or hydrolases. The results indicated that O-glycosylation was indeed limiting in the expression of recombinant hCG protein, and N-acetylgalactosamine (GalNAc) was the major limiting precursor. Glutamine-fructose-6-phosphate transaminase 2 (Gfat2) and Uridine diphosphate-glucose pyrophosphorylase 2 (Ugp2), key regulatory genes of O-glycan precursor synthesis, were overexpressed. Ugp2 overexpression significantly increased the recombinant hCG protein level by 1.92 times compared to that of the control. The LC-MS/MS analysis and Phaseolus vulgaris leucoagglutinin (PHA-L) lectin blot analysis showed that Ugp2 overexpression significantly increased the total galactosylation levels of intracellular proteins and the O-glycosylation of recombinant hCG protein. The stability of the hCG protein to trypsin digestion was also enhanced. Ugp2 is the major limiting enzyme of the O-glycan precursor synthesis in recombinant hCG protein production. Furthermore, the effects and mechanisms of the key genes of O-glycan sugar chain synthesis and hydrolases such as polypeptide N-acetylgalactosaminyltransferase1 (Galnt1), Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase (C1galt1), O-linked N-acetylglucosamine transferase (Ogt) and Hexosaminidase (Hex), were evaluated. The results indicated that Galnt1 overexpression increased the recombinant hCG protein level by 1.57 times and improved the total galactosylation of intracellular proteins, O-glycosylation and the stability of recombinant hCG protein. Galnt1 is the major limiting enzyme of O-glycan sugar chain synthesis. Overexpression of Ugp2 and Galnt1 simultaneously improved the recombinant hCG protein level by 2.44 times, and both had synergistic effects. Based on the results of overexpression of Galnt1, the major limiting gene of O-Glycan chain synthesis, the precursors GalNAc and Gal were added and increased the recombinant hCG protein level by 3.68 times. This study revealed the major limiting factors of O-glycosylation of recombinant hCG protein in CHO cells and proposed an effective expression regulation strategy.