A kind of n-glycosyltransferase mutant p1 and its application

Abstract

The invention discloses an N-glycosyltransferase mutant P1, which is obtained from the N-glycosyltransferase gene derived from Actinobacillus pleuropneumoniae through three point mutations of S33A, M158L and K203Q; its amino acid sequence is as shown in SEQ ID Shown in NO.1. The present invention also discloses the application of the mutant P1 as a tool enzyme for polypeptide glycosylation modification in vivo in utilizing UDP-Glc to glycosylate polypeptides containing N-X-S / T sequences to form glycopeptides or glycoproteins . The mutant P1 of the present invention has completely lost the ability to utilize UDP-Gal, but still retains the ability to utilize UDP-Glc for glycosylation, and can be used to construct an Escherichia coli expression system or a Bacillus subtilis expression system using UDP in microorganisms ‑Glc produces glycopeptides or glycoproteins with a uniform Glc, avoiding the use of the more expensive substrate UDP‑Glc. This provides a new way for the glycosylation modification of polypeptides and proteins, and provides a convenient method for the synthesis of glycoprotein vaccines.

Description

technical field [0001] The present invention relates to a kind of glycosyltransferase and its application, especially to a kind of N-glycosyltransferase mutant (named P1) derived from Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) and its application, belonging to molecular Technical field of glycoengineering in biology. Background technique [0002] The glycosylation modification of protein has a great impact on the function and physicochemical properties of protein. Proteins synthesized via the ER pathway are mostly modified with N-linked glycans. N-linked glycans can promote correct protein folding, improve protein water solubility, and regulate drug protein metabolic half-life. [0003] The current method of producing glycoproteins by chemical synthesis or in vivo extraction is expensive and complicated. NGT can directly add free UDP-Glc / UDP-Gal to the peptide sequence with N-X-S / T. However, when NGT is used to synthesize glycopeptides or glycoprote...

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Problems solved by technology

[0003] The current method of producing glycoproteins by chemical synthesis or in vivo extraction is expensive and complicated

NGT can directly add free UDP-Glc / UDP-Gal to the peptide sequence with N-X-S / T. However, when NGT is used to synthesize glycopeptides or glycoproteins in vitro, the cost of UDP-Glc and UDP-Gal is relatively high, and the use of large intestine When Bacillus or Bacillus subtilis expression system expresses NGT in vivo to synthesize glycopeptides or glycoproteins, the sugar donor recognized by NGT is not specific, resulting in the inability to synthesize glycopeptides or glycoproteins with uniform Glc

After searching, it has completely lost the ability to use UDP-Gal, but still retains the ability to use UDP-Glc for glycosylation, and can be transferred into E. coli or Bacillus subtilis as an in vivo glycosylation system to produce a homogeneous Glc The specificity of glycopeptide-enhanced N-glycosyltransferases has not been reported

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