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Target protein expression with laetiporus sulphureus mushroom lectin N-acetyllactosamine binding domain as fusion tag and purification method thereof

A technology of acetyllactosamine and fusion protein, which is applied in the field of genetic engineering, can solve the problems of affecting gene expression level, protein insolubility, low expression of tagged protein and target protein, and achieve the effect of reducing purification cost

Active Publication Date: 2016-02-03
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In addition, because each organism has a large difference in the utilization of codons when translating genes, this difference directly affects the level of gene expression
As a fungal protein, mushroom lectin has a low codon utilization rate in E. coli, which will directly lead to low expression levels of subsequent tagged proteins and target proteins, and easily lead to insoluble expressed proteins.

Method used

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  • Target protein expression with laetiporus sulphureus mushroom lectin N-acetyllactosamine binding domain as fusion tag and purification method thereof
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  • Target protein expression with laetiporus sulphureus mushroom lectin N-acetyllactosamine binding domain as fusion tag and purification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1: the synthesis of LSL gene and the construction of label gene

[0060] The DNA analysis and RNA structure prediction of the LSLa gene (its nucleotide sequence is shown in SEQ ID NO.3) encoding the N-acetyllactosamine binding domain of the sulfur bacteria mushroom lectin was artificially synthesized without changing the natural amino acid sequence The LSL gene encoding the N-acetyllactosamine binding domain of the sulfur bacteria mushroom lectin, the nucleotide sequence of the artificially synthesized LSL gene is the nucleotide sequence shown in SEQ ID NO.1.

[0061]Add the NcoI restriction endonuclease recognition site at the 5' end of the above-mentioned artificially synthesized LSL gene sequence, and add the KpnI restriction endonuclease recognition site, TEV protease recognition site and The BamHI restriction endonuclease recognizes the site to form a tag gene, and then clones the tag gene into the pET32a(+) plasmid to obtain the pET32a-LSL plasmid conta...

Embodiment 2

[0062] Example 2: Construction of an expression vector containing the LSL gene encoding the sulfur bacteria mushroom lectin N-acetyllactosamine binding domain

[0063] 1. Construction of pET28a-LSL expression vector

[0064] The pET32a-LSL plasmid containing the tag gene synthesized in Example 1 was double digested with NcoI and BamHI, and the tag gene fragment containing both the LSL gene and the TEV protease recognition site was recovered by agarose gel electrophoresis; the recovery was obtained by T4DNA ligase The tag gene fragment containing both the LSL gene and the TEV protease recognition site was connected to the pET28a(+) expression vector after double digestion with NcoI and BamHI; then the ligation product was transformed into E. coli competent cells DH5α, and The transformed DH5α was spread onto the LB solid culture plate containing kanamycin and cultured overnight at 37°C. The next day, a single colony was picked and placed in 5ml of LB liquid medium containing k...

Embodiment 3

[0074] Embodiment 3: Expression of the fusion protein containing LSL protein tag

[0075] 1. Expression of fusion protein containing LSL protein tag and Cap protein (LSL-Cap fusion protein for short)

[0076] Expression method of fusion protein: Transform the expression vector pET28a-LSL-Cap into E.coliBL21(DE3) host bacteria, and then apply the transformed E.coliBL21(DE3) to the LB solid culture plate containing kanamycin , overnight at 37°C. The next day, pick a single colony and place it in 5ml LB liquid medium containing kanamycin, and culture it at 200rpm at 37°C for 12h; At 37°C, 200rpm shaking culture until the OD600≈0.8 of the bacterial liquid, adding isopropylthiogalactopyranoside (IPTG) with a final concentration of 1.0mmol / L, and inducing culture at 25°C, 200rpm for 6h. Centrifuge the cultured bacterial solution at 8000rpm for 5 minutes, discard the supernatant, collect the bacterial cell pellet, and add bacterial cell pellet with bacterial cell pellet to resuspen...

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Abstract

The invention discloses an artificially-synthesized LSL gene for encoding a laetiporus sulphureus mushroom lectin N-acetyllactosamine binding domain and an expression vector and host cell containing the LSL gene. The nucleotide sequence of the LSL gene is SEQ ID NO. 1. The invention further discloses a target protein expression with LSL used as the fusion tag and a purification method thereof. The method specifically relates to LSL gene synthesis, construction of the expression vector containing the LSL gene and the target protein gene, construction of the expression vector containing the LSL gene and a protease gene, expression and purification of fusion protein, LSL protein tag removal, target protein purification and the like. The method is simple and easy to implement, one-step purification of target protein is achieved, high-purity target protein can be obtained, the protein purification cost is lowered, the method can be widely applied to activity protein in the fields of biological medicine, veterinary medicine and the like and large-scale preparation of vaccine antigen in the industry of biological products, and high practical value is achieved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for expressing and purifying a target protein using the sulfur bacteria mushroom lectin N-acetyllactosamine binding domain as a fusion tag. Background technique [0002] With the development of genetic engineering technology, genomics and proteology technology, recombinant protein has been widely used in various fields such as biology, medicine, agriculture and animal husbandry and veterinary medicine. However, large-scale production and purification of proteins with exact properties are still difficult points in recombinant protein technology. There are two types of expression systems for recombinant proteins: prokaryotic and eukaryotic. Eukaryotic expression systems include yeast, insect and mammalian cell expression systems, etc. Compared with the tedious process and high cost of eukaryotic expression system, prokaryotic expression system rep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N1/21C07K1/16
Inventor 赵军王川庆高冬生杨霞常洪涛陈陆王新卫李永涛刘红英姚慧霞李晓静
Owner HENAN AGRICULTURAL UNIVERSITY
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