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A kind of n-glycosyltransferase mutant f13 and its application

A technology of glycosyltransferase and mutant, applied in the directions of transferase, enzyme, hydrolase, etc., can solve the problems of incapable of glycosylation of polypeptides, unreported N-glycosyltransferase, complicated steps, etc.

Active Publication Date: 2022-03-22
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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.

Method used

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  • A kind of n-glycosyltransferase mutant f13 and its application
  • A kind of n-glycosyltransferase mutant f13 and its application
  • A kind of n-glycosyltransferase mutant f13 and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Construction of N-glycosyltransferase ApNGT mutant F13 and expression, purification and identification of F13 protein

[0029] 1. Construction of expression strains

[0030] In the early stage, a large number of NGT site-directed mutation strains were constructed and mutant F13 was screened. The N-glycosyltransferase gene in wild-type N-glycosyltransferase (NCBI Reference Sequence: WP_005605627.1) Actinobacillus pleuropneumoniae was synthesized by Nanjing GenScript and constructed on the vector pET45b, which is commercialized The strain is DH5α-pET45b-ApNGT strain.

[0031] Use the DH5α-pET45b-ApNGT strain, culture the DH5α-pET45b-ApNGT strain in advance with 0.1 mg / mL LB liquid medium at 170 rpm at 37°C to OD 600 Reach 0.6, and mention the plasmid as a template.

[0032] According to Novizym one-step multi-point mutagenesis kit (C25-01) instructions, combined with Novizym online primer design program CE Design to design mutagenic primers (see Table 2). N...

Embodiment 2

[0040] Example 2: Application of N-glycosyltransferase mutant F13 in polypeptide glycosylation modification

[0041] Using UDP-Glc / UDP-Gal / UDP-GlcA as sugar donors, react with 20uL of the reaction system shown in Table 3 at 37°C for 3h, heat at 100°C for 10min after the reaction, centrifuge at 12000rpm for 10min, take the supernatant and dilute 100 times, used for ESI-MS detection. Among them, the substrate peptides used for experimental verification are shown in Table 4.

[0042] Table 3: Reaction system.

[0043]

[0044] Table 4: Substrate peptides used for experimental validation

[0045]

[0046] The enzyme activity results of F13 protein showed that the F13 mutant could not only glycosylate the natural substrate HMW1 fragment of wild-type NGT ( figure 2 ), and can glycosylate the fragment of diformyl peptidase CDTPANCTYLDLL ( image 3 ) and hemagglutinin fragment TLDDNGTMLFFK ( Figure 4 ) and IgG fragment REEQYNSTYRVVS ( Figure 5 ), indicating that the mut...

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Abstract

The invention discloses an N-glycosyltransferase mutant F13, which is obtained from an N-glycosyltransferase gene derived from Actinobacillus pleuropneumoniae through three-point mutation of N471I, S275A and P497R; its amino acid sequence is as shown in SEQ ID NO.1 is shown. The invention also discloses the application of the mutant F13 as a tool enzyme for polypeptide glycosylation modification to glycosylate a polypeptide containing N-X-S / T sequence to form a glycopeptide or a glycoprotein. The mutant F13 of the present invention has significantly improved glycosylation efficiency and substrate range compared to wild-type NGT, can extensively glycosylate fragments of wild-type NGT that cannot be glycosylated such as IgG fragments and can utilize sugar donors that cannot be used by wild-type UDP-GlcA can be used to easily and quickly use UDP-Glc / UDP-Gal / UDP-GlcA to glycosylate peptides containing N-X-S / T sequences, and further use other glycosyltransferases to The sugar chain of the glycopeptide is modified to obtain the target glycopeptide. This provides a new way for the glycosylation modification of polypeptides and proteins, and provides a simple method for the 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12P21/02C12N9/48
CPCC12N9/1051C12P21/005C12P21/02C12N9/485
Inventor 陈敏李昆刘昭曦
Owner SHANDONG UNIV
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