CN113249353A

CN113249353A - N-glycosyltransferase mutant F13 and application thereof

Info

Publication number: CN113249353A
Application number: CN202110527726.0A
Authority: CN
Inventors: 陈敏; 李昆; 刘昭曦
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2021-05-14
Filing date: 2021-05-14
Publication date: 2021-08-13
Anticipated expiration: 2041-05-14
Also published as: CN113249353B

Abstract

The invention discloses an N-glycosyltransferase mutant F13, which is obtained by carrying out three-point mutation on an N-glycosyltransferase gene from actinobacillus pleuropneumoniae, namely N471I, S275A and P497R; the amino acid sequence is shown in SEQ ID NO. 1. The invention also discloses application of the mutant F13 as a tool enzyme for polypeptide glycosylation modification to glycosylate the polypeptide containing the N-X-S/T sequence to form glycopeptide or glycoprotein. Compared with wild NGT, the mutant F13 of the invention has obviously improved glycosylation efficiency and substrate range, can widely glycosylate fragments which cannot be glycosylated by wild NGT, such as IgG fragments, and can utilize a sugar donor UDP-GlcA which cannot be utilized by wild NGT, can simply and quickly carry out glycosylation modification on a polypeptide containing an N-X-S/T sequence by utilizing UDP-Glc/UDP-Gal/UDP-GlcA, and can further utilize other glycosyltransferases to modify the sugar chain of the glycopeptide to obtain the target glycopeptide. The method provides a new way for glycosylation modification of polypeptide and protein, and provides a simple method for synthesis of glycoprotein vaccine.

Description

N-glycosyltransferase mutant F13 and application thereof

Technical Field

The invention relates to a glycosyltransferase and application thereof, in particular to a mutant (named F13) of N-glycosyltransferase from Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) and application thereof, belonging to the technical field of sugar engineering in molecular biology.

Background

Glycosylation modification of proteins has a great influence on the functions and physicochemical properties of proteins, and most antibodies and cytokines are N-glycosylated. Inhibition of protein glycosylation often affects protein proper folding, metabolism, and protein function. Researches also find that the glycoprotein vaccine formed by combining the sugar and the carrier protein can stimulate the body to generate immune memory, so that the research of the glycoprotein vaccine draws extensive attention. The glycoprotein is obtained by directly extracting in vivo or chemically synthesizing in vitro, and the two methods have the defects of complicated steps and high cost.

NGT derived from Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) has been reported to be capable of glycosylating polypeptides having the sequence N-X-S/T (X.noteq.Pro) directly using free UDP-Glc and UDP-Gal, with simple steps and low cost, but wild-type NGT cannot glycosylate all polypeptides having the sequence N-X-S/T (X.noteq.Pro), such as Fc fragments of IgG, and cannot use UDP-GlcA as a sugar donor. The glycosylation efficiency is improved in the mutations of ApNGT reported so far, but no mutants capable of glycosylating an Fc fragment of IgG, and N-glycosyltransferase and mutants capable of using UDP-GlcA as a donor have been reported.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an N-glycosyltransferase mutant derived from Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) and application thereof in N-linked glycosylation modification of polypeptides.

The N-glycosyltransferase mutant is obtained by carrying out three-point mutation on an N-glycosyltransferase gene which is derived from Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) and is NCBI Reference Sequence WP-005605627.1 through N471I, S275A and P497R; the method is characterized in that: the mutant is named as N-glycosyltransferase mutant F13, the amino acid sequence of the mutant is shown in SEQ ID NO.1, the glycosylation efficiency and the substrate range of the mutant are obviously improved compared with wild type NGT, the mutant can be used for widely glycosylating the fragment which cannot be glycosylated by the wild type NGT and can utilize a sugar donor UDP-GlcA which cannot be utilized by the wild type.

The N-glycosyltransferase mutant F13 is obtained by constructing an N-glycosyltransferase gene in wild-type N-glycosyltransferase (NCBI Reference Sequence: WP _005605627.1) Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) on a vector pET45b, constructing a mutant F13 (three-point mutation of N471I, S275A and P497R) by using a Novozan one-step method multi-point mutation kit (C25-01), correctly sequencing by a biological engineering company Limited, fermenting, inducing, expressing, purifying and recovering the Escherichia coli BL 21.

The invention also provides application of the N-glycosyltransferase mutant F13 in N-linked glycosylation modification of a polypeptide.

Furthermore, the N-glycosyltransferase mutant F13 provided by the invention is used as a tool enzyme for in vivo polypeptide glycosylation modification to glycosylate a polypeptide containing an N-X-S/T sequence to form a glycopeptide or glycoprotein by using UDP-Glc or UDP-Gal or UDP-GlcA.

Wherein: compared with the wild NGT, the glycosylation efficiency and the substrate recognition range of the N-glycosyltransferase mutant F13 serving as a tool enzyme are remarkably improved, and Glc/Gal/GlcA on UDP-Glc or UDP-Gal or UDP-GlcA can be transferred to a wide range of polypeptides containing N-X-S/T sequences to form glycopeptides; further modification of the sugar chain of a glycopeptide with other glycosyltransferases can be used to produce a pharmaceutical protein with glycosylation modifications or a glycopeptide of interest.

Wherein: the pharmaceutical protein is preferably an antibody, cytokine or glycoprotein vaccine.

The N-glycosyltransferase mutant F13 disclosed by the invention belongs to an important glycosyltransferase modified by N-glycosylation, not only has obviously improved glycosylation efficiency compared with the wild ApNGT, but also can be used for glycosylating peptide substrates which cannot be glycosylated by the wild ApNGT, such as hemagglutinin fragments and IgG fragments, and can be used for taking UDP-GlcA as a sugar donor. After production of a glycopeptide having Glc/Gal/GlcA, the sugar chain of the glycopeptide may be further modified with another glycosyltransferase to obtain the desired glycopeptide. The method provides a new way for glycosylation modification of polypeptide and protein, and provides a simple method for synthesis of glycoprotein vaccines.

Drawings

FIG. 1: SDS-PAGE Coomassie blue staining of F13 protein.

Wherein: m represents protein Maker, and F13 has molecular weight of about 70 KDa.

FIG. 2: the results of wild-type ApNGT and F13 mutants on the enzymatic activity of HWM fragments are shown.

The donor was UDP-Glc, substrate 562.24, product 643.27, wherein: ApNGT; f13 mutants.

FIG. 3: the results of wild-type ApNGT and F13 mutants on the enzymatic activity of the dicarboxyl peptidase fragments are shown.

The donor is UDP-Glc, substrate 731.31, product 812.33 wherein: ApNGT; f13 mutants.

FIG. 4: the results of wild-type ApNGT and F13 mutant on the enzyme activity of hemagglutinin fragment are shown.

The donor was UDP-Glc, substrate 751.86, product 832.89. Wherein: ApNGT; f13 mutants.

FIG. 5: the results of wild-type ApNGT and F13 mutants on the enzyme activity of the IgG fragment are shown.

The donor was UDP-Glc, and the substrates were 544.27 and 544.60([ M +3H)/3) and 815.90 and 816.40([ M +3H ]/3).

Wherein: ApNGT; f13 mutants.

FIG. 6: the F13 mutant utilizes UDP-GlcA enzyme activity result chart.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.

In the following examples, materials, reagents and the like used were obtained commercially unless otherwise specified. The sources of reagents and consumables involved are shown in table 1.

TABLE 1 details of sources of reagents and consumables to which the examples relate

Example 1: construction of N glycosyltransferase ApNGT mutant F13, expression, purification and identification of F13 protein

1. Construction of expression Strain

The mutant F13 is obtained by constructing NGT site-directed mutant strain in large quantity and screening. N-glycosyltransferase gene of wild-type N-glycosyltransferase (NCBI Reference Sequence: WP-005605627.1) Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) was synthesized by Kinshire, Nanjing and constructed on vector pET45b, which is a commercially available strain of DH5 alpha-pET 45 b-ApNGT.

Using DH5 alpha-pET 45b-ApNGT strain, DH5 alpha-pET 45b-ApNGT strain was cultured to OD at 37 ℃ in advance with LB liquid medium containing 0.1mg/mL at 170rpm₆₀₀Up to 0.6 and the upgraded grain serves as a template.

Mutagenesis primers were designed according to the Novozan one-step multiple-point mutation kit (C25-01) in combination with the Novozan on-line primer Design procedure CE Design (see Table 2). Three-point mutation of N471I, S275A and P497R of N-glycosyltransferase gene derived from Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) to obtain N-glycosyltransferase mutant.

Table 2: primers required for construction of F13 mutant

And (3) using a mutagenic primer to amplify the fragment carrying the mutation in a segmented way, using agarose gel electrophoresis to determine the correctness of the size of the amplified fragment, and then carrying out Dpn I enzyme digestion to remove the methylated template plasmid. And then carrying out recombinant cyclization on the fragments carrying the mutation according to a recombinant cyclization system of the kit, transferring the recombinant fragments into DH5 alpha competent cells after recombination, coating an ampicillin solid LB plate containing 0.1mg/mL, culturing for 16h, selecting clones, and handing over the clones to Shanghai biological engineering company for sequencing, determining the mutants to be N-glycosyltransferase after the sequencing is correct (named as F13, and the amino acid sequence of the mutants is shown as SEQ ID NO. 1), and transferring the re-upgraded particles into BL21(DE3) competent cells for expression.

2. Expression, purification and identification of F13 protein

After the F13 mutant was transferred to BL21 competent cells, the cells were inoculated into 1L of LB liquid medium containing 0.1mg/mL ampicillin, and cultured at 37 ℃ and 200rpm to OD₆₀₀To reach 0.6, IPTG was added to a final concentration of 0.2mM and incubated at 16 ℃ for 20 h. After the culture was completed, the cells were collected by centrifugation at 8000rpm for 10 min. The cells were resuspended in 50mL PBS and sonicated for 30min at 200W for 2s with 2s pause in an ice water environment to disrupt cell-released proteins, after which the pellet was separated by centrifugation at 12000rpm for 20min and the supernatant was purified on a Ni-packed column.

After passing the supernatant through a Ni-packed column, the impure proteins were eluted using 10 column volumes Washing buffer. Then eluting with 10 column volumes of Elution buffer and collecting the target protein. And adding the collected eluent into an ultrafiltration tube, centrifuging for 30min each time, discarding tube liquid after centrifugation, filling PBS into the upper tube, repeatedly centrifuging for several times to remove imidazole, and storing the obtained target protein at 4 ℃.

Protein concentration was determined according to the instructions of the industrial BAC protein concentration determination kit. Adding SDS-PAGE loading buffer solution into the sample, boiling for 10min, loading the sample into 12% SDS-PAGE gel, performing electrophoresis at 130V for 90min, staining for 2h, and decolorizing. The results of SDS-PAGE Coomassie blue staining of the F13 protein are shown in FIG. 1.

Example 2: application of N-glycosyltransferase mutant F13 in polypeptide glycosylation modification

UDP-Glc/UDP-Gal/UDP-GlcA was used as a sugar donor, reacted at 37 ℃ for 3 hours in a reaction system shown in Table 3 of 20uL, heated at 100 ℃ for 10min after the reaction was completed, centrifuged at 12000rpm for 10min, and the supernatant was diluted 100 times and used for ESI-MS detection. Among them, the substrate peptides used for experimental verification are shown in table 4.

Table 3: and (3) reaction system.

Table 4: substrate peptides for experimental validation

The results of the enzyme activities of the F13 protein show that the F13 mutant is capable of glycosylating not only the HMW1 fragment, which is the natural substrate of wild-type NGT (fig. 2), but also the CDTPANCTYLDLL (fig. 3) and the hemagglutinin TLDDNGTMLFFK (fig. 4) and the IgG REEQYNSTYRVVS (fig. 5), which are fragments of diformylpeptidase that the wild-type NGT is not glycosylated, indicating that the mutant F13 has extensive glycosylation capability and can be used for producing antibodies with glycosylation. And the F13 mutant can use UDP-GlcA that wild-type NGT cannot use as a donor (fig. 6), indicating that F13 can be used as an engineered enzyme for producing glycopeptides with GlcA.

Sequence listing

<110> Shandong university

<120> N glycosyltransferase mutant F13 and application thereof

<141>2021-5-13

<160>1

<210>1

<211> 620

<212>PRT

<213> Artificial sequence

<221> amino acid sequence of N glycosyltransferase mutant F13

<222>（1）…（620）

<400>1

MENENKPNVA NFEAAVAAKD YEKACSELLL ILSQLDSNFG GIHEIEFEYP AQLQDLEQEK 60

IVYFCTRMAT AITTLFSDPV LEISDLGVQR FLVYQRWLAL IFASSPFVNA DHILQTYNRE 120

PNRKNSLEIH LDSSKSSLIK FCILYLPESN VNLNLDVMWN ISPELCASLC FALQSPRFVG 180

TSTAFNKRAT ILQWFPRHLD QLKNLNNIPS AISHDVYMHC SYDTSVNKHD VKRALNHVIR 240

RHIESEYGWK DRDVAHIGYR NNKPVMVVLL EHFHAAHSIY RTHSTSMIAA REHFYLIGLG 300

SPSVDQAGQE VFDEFHLVAG DNMKQKLEFI RSVCESNGAA IFYMPSIGMD MTTIFASNTR 360

LAPIQAIALG HPATTHSDFI EYVIVEDDYV GSEECFSETL LRLPKDALPY VPSALAPEKV 420

DYLLRENPEV VNIGIASTTM KLNPYFLEAL KAIRDRAKVK VHFHFALGQS IGITHPYVER 480

FIKSYLGDSA TAHPHSRYHQ YLRILHNCDM MVNPFPFGNT NGIIDMVTLG LVGVCKTGAE 540

VHEHIDEGLF KRLGLPEWLI ANTVDEYVER AVRLAENHQE RLELRRYIIE NNGLNTLFTG 600

DPRPMGQVFL EKLNAFLKEN 620

Claims

1. An N-glycosyltransferase mutant is obtained by carrying out three-point mutation on an N-glycosyltransferase gene which is derived from Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) and is NCBI Reference Sequence WP _005605627.1 through N471I, S275A and P497R; the method is characterized in that: the mutant is named as N-glycosyltransferase mutant F13, the amino acid sequence of the mutant is shown in SEQ ID NO.1, the glycosylation efficiency and the substrate range of the mutant are obviously improved compared with wild type NGT, the mutant can be used for widely glycosylating the fragment which cannot be glycosylated by the wild type NGT and can utilize a sugar donor UDP-GlcA which cannot be utilized by the wild type.

2. Use of the N-glycosyltransferase mutant of claim 1 as a tool enzyme for the in vivo modification of polypeptide glycosylation to glycosylate a polypeptide comprising an N-X-S/T sequence with UDP-Glc or UDP-Gal or UDP-GlcA to form a glycopeptide or glycoprotein.

3. Use according to claim 2, characterized in that: compared with the wild NGT, the glycosylation efficiency and the substrate recognition range of the N-glycosyltransferase mutant F13 serving as a tool enzyme are remarkably improved, and Glc/Gal/GlcA on UDP-Glc or UDP-Gal or UDP-GlcA can be transferred to a wide range of polypeptides containing N-X-S/T sequences to form glycopeptides; further modification of the sugar chain of a glycopeptide with other glycosyltransferases can be used to produce a pharmaceutical protein with glycosylation modifications or a glycopeptide of interest.

4. Use according to claim 3, characterized in that: the pharmaceutical protein is an antibody, cytokine or glycoprotein vaccine.