N glycosyl transferase mutant F13 and application thereof

Abstract

The invention discloses an N glycosyl transferase mutant F13. The N glycosyl transferase mutant F13 is obtained by carrying out N471I, S275A and P497R three-point mutation on an N-glycosyl transferase gene from actinobacillus pleuropneumoniae; the amino acid sequence thereof is as shown in SEQ ID NO. 1. The invention also discloses application of the mutant F13 as a tool enzyme for polypeptide glycosylation modification in glycosylation of a polypeptide containing an N-X-S / T sequence to form glycopeptide or glycoprotein. Compared with wild type NGT, the mutant F13 disclosed by the invention has the advantages that: the glycosylation efficiency and the substrate range are remarkably improved; fragments, such as IgG fragments, which cannot be glycosylated by the wild type NGT can be widely glycosylated; furthermore, a sugar donor UDP-GlcA which cannot be utilized by the wild type can be utilized; the polypeptide containing the N-X-S / T sequence can be simply and quickly subjected to glycosylation modification by utilizing UDP-Glc / UDP-Gal / UDP-GlcA; and other glycosyl transferase can be further utilized to modify the sugar chain of the glycopeptide to obtain the target glycopeptide. A new way is provided for glycosylation modification of polypeptide and protein; and a simple and convenient method is provided for synthesis of glycoprotein vaccines.

Description

technical field [0001] The present invention relates to a kind of glycosyltransferase and its application, in particular to a kind of N-glycosyltransferase mutant (named as F13) 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 influence on the function and physicochemical properties of protein. Most antibodies and cytokines are N-glycosylated. Inhibition of protein glycosylation often affects proper protein folding, metabolism, and protein function. Some studies have also found that combining sugars with carrier proteins to form glycoprotein vaccines can stimulate the body to produce immune memory, so the research on glycoprotein vaccines has attracted widespread attention. In the past, glycoproteins were obtained through direct extraction in vivo, or chemical synthesis ...

AI Technical Summary

Problems solved by technology

In the past, glycoproteins were obtained through direct extraction in vivo, or chemical synthesis in vitro, both of which have the disadvantages of complicated steps and high cost.

[0003] The reported NGT derived from Actinobacillus pleuropneumoniae can directly use free UDP-Glc and UDP-Gal to glycosylate polypeptides with N-X-S / T (X≠Pro) sequence, with simple steps and low cost. However, wild-type NGT cannot glycosylate all polypeptides with the N-X-S / T (X≠Pro) sequence, such as the Fc fragment of IgG, and cannot use UDP-GlcA as a sugar donor

The glycosylation efficiency has been improved in the mutation of ApNGT that has been reported so far, but the mutants that can glycosylate the Fc fragment of IgG and the N-glycosyltransferases and mutants that can use UDP-GlcA as a donor have not been reported.

Images