Prokaryotic Expression and Activity Identification of Human Alpha-1,3-Mannosyl-Glycoprotein 2-Beta-N-Acetylglucosaminyltransferase (hGnT-I)
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Q819

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    Abstract:

    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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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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History
  • Online: October 27,2021
  • Published: October 25,2021
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