人源β-N-乙酰葡糖胺转移酶I（h-GnT-I）的原核表达与活性鉴定

doi:10.3969/j.issn.1673-1689.2021.10.008

首页 > 过刊浏览>2021年第40卷第10期 >56-62. DOI:10.3969/j.issn.1673-1689.2021.10.008

人源β-N-乙酰葡糖胺转移酶I（h-GnT-I）的原核表达与活性鉴定
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                        10.3969/j.issn.1673-1689.2021.10.008
                    
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Prokaryotic Expression and Activity Identification of Human Alpha-1,3-Mannosyl-Glycoprotein 2-Beta-N-Acetylglucosaminyltransferase (hGnT-I)

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β-N-乙酰葡糖胺转移酶I（h-GnT-I）功能为在高尔基体中向糖蛋白上的N-寡糖M5GN2结构添加一个β--1，2 连接的N-乙酰葡糖胺。为大量获得具有活性的h-GnT-I蛋白，构建了重组表达质粒pET28a-MGAT1ΔTM并将其在大肠杆菌Rosetta菌株中表达。对诱导条件进行优化后，将获得的可溶性重组蛋白His-hGnT-IΔTM进行镍柱亲和纯化并通过高效液相色谱（HPLC）检测其体外活性和底物特异性。结果表明，大肠杆菌ROSETTA体系可以成功表达可溶性重组hGnT-I蛋白；该蛋白质相对分子质量约为42 800，在体外反应中具有相应的糖基转移酶活性；除天然糖链底物M5GN2外，该蛋白质可以识别非天然糖链底物M3GN2；产物经酶切反应验证，证明该蛋白质催化添加一个β糖苷键连接的N-乙酰葡糖胺的反应。本研究所开发的原核表达体系可以大量制备纯化的功能性重组hGnT-I蛋白，以应用于该蛋白质的性质研究以及N-糖链的体外合成。

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Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase(hGnT-I) works in Golgi glycosylation pathway and adds a β-1,2 linked GlcNAc to Man5Gn2 structure on glycoproteins. The recombinant plasmid pET28a-MGAT1ΔTM was constructed and expressed in the Escherichia coli Rosetta strain to obtain the active hGnT-I in large quantity. After optimizing the induction conditions, the soluble recombinant protein His-hGnT-IΔTM was successfully obtained and then purified by Ni-NTA column. The in vitro activity assay and substrate specificity detected by high-performance liquid chromatography(HPLC). The results suggested that the recombinant protein hGnT-IΔTM with the molecular weight of 42 800 could be successfully expressed in the ROSETTA system of E. coli, showing the corresponding glycosyltransferase activity in vitro. Except for its natural substrate M5GN2, hGnT-IΔTM also showed comparable glycosyltransferase activity against the non-natural substrate M3GN2. The products were verified by enzymatic digestion with β-N-actyl-glucosaminidase, indicating that the protein catalyzed the addition of a β glycoside bond linked to N-acetyl-glucosamine in the reaction. The prokaryotic expression system developed in this study could be used to produce a large number of purified functional recombinant hGnT-IΔTM, which could be used to the further investigation of this protein and the in vitro chemo-enzymatic synthesis of N-glycans.

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陆天天,王宁,高晓冬.人源β-N-乙酰葡糖胺转移酶I（h-GnT-I）的原核表达与活性鉴定[J].食品与生物技术学报,2021,40(10):56-62.

LU Tiantian, WANG Ning, GAO Xiaodong. Prokaryotic Expression and Activity Identification of Human Alpha-1,3-Mannosyl-Glycoprotein 2-Beta-N-Acetylglucosaminyltransferase (hGnT-I)[J]. Journal of Food Science and Biotechnology,2021,40(10):56-62.

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在线发布日期: 2021-10-27
出版日期: 2021-10-25

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