| 论文题目: | Expression of glycoproteins bearing complex human-like glycans with galactose terminal in Hansenula polymorpha |
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| 作者: | Wang Hui, Song Hao-Lei, Wang Qian, Qiu Bing-Sheng |
| 联系作者: | Qiu Bing-Sheng |
| 刊物名称: | World journal of microbiology & biotechnology |
| 期: | 3 |
| 卷: | 29 |
| 页: | 447-58 |
| 年份: | 2013 |
| 影响因子: | 1.602 |
| 论文下载: | |
| 摘要: | Glycoproteins derived from Hansenula polymorpha can not be used for therapeutic purposes due to their high-mannose type asparagine-linked (N-linked) glycans, which result in immune reactions and poor pharmacokinetic behaviors in human body. Previously, we reported that the trimannosyl core N-linked glycans (Man(3)GlcNAc(2)) intermediate can be generated in endoplasmic reticulum in HpALG3 and HpALG11 double-mutant H. polymorpha. Here, we describe the further modification of the glycosylation pathway in this double-defect strain to express glycoproteins with complex human-like glycans. After eliminating the impact of HpOCH1, three glycosyltransferases were introduced into this triple-mutant strain. When human beta-1,2-N-acetylglucosaminyltransferase I (hGnTI) was efficiently targeted in early Golgi, more than 95 % glycans attached to the glycoproteins were added one N-acetylglucosamine (GlcNAc). With subsequently introduction of rat beta-1,2-N-acetylglucosaminyltransferase II (rGnTII) and human beta-1,4-galactosyltransferase I (hGalTI), several glycoengineered strains can produce glycoproteins bearing glycans with terminal N-acetylglucosamine or galactose. The expression of glycoproteins with glycan Gal(2)GlcNAc(2)Man(3)GlcNAc(2) represents a significant step toward the ability to express fully humanized glycoproteins in H. polymorpha. Furthermore, several shake-flask and bioreactor fermentation experiments indicated that, although the cells do display a reduction in growth rate, the glycoengineered strains are still suitable for high-density fermentation. |
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