Method and application of heterologous high expression of dypb active protein

An active protein, high expression technology, applied in the field of heterologous high expression of DypB active protein, can solve problems such as increasing product cost, reducing production efficiency, increasing production links, etc. Effect

Active Publication Date: 2020-05-05
四川爱奇生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzyme can be recombinantly expressed in E.coli, but it has the activity of degrading lignin only after being combined with porphyrin in vitro, which increases the production process, increases the product cost, and reduces the production efficiency

Method used

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  • Method and application of heterologous high expression of dypb active protein
  • Method and application of heterologous high expression of dypb active protein
  • Method and application of heterologous high expression of dypb active protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of BL21-AL expression host

[0046] (1) Construction of recombinant vector pET28a-hemAL-cm

[0047] The hemA gene and hemL gene of Bacillus subtilis were retrieved through NCBI, the codons were optimized, and the rare codons expressed by E.coli were removed through synonymous mutation, and the hemA gene and nucleotide sequence with the nucleotide sequence of Seq ID No.1 were obtained It is the hemL gene of Seq ID No.2, two genes are connected in series, (G 4 S)x2 is linker, tac promoter to start expression, 36bp homology arm at the 3rd end of thrA gene whose nucleotide sequence is Seq ID No.11 is added to the hemL end, and finally enzyme cutting sites (BglⅡ) and (EcoRI) are added at both ends It was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The synthetic sequence was inserted into pET28a plasmid via (BglⅡ) and (EcoRI) to construct pET28a-hemAL.

[0048] Using the plasmid pkd3 template, using the nucleotide sequence as the primer 1...

Embodiment 2

[0051] Example 2 Construction of pCspA-dypB expression vector

[0052] The dypB gene is derived from Rhodococcus jostii RHA1. The dypB gene sequence of Rhodococcus jostii RHA1 was retrieved through NCBI, the codons were optimized, and enzyme cutting sites (NdeⅠ) and (HindⅢ) were added at both ends, and delivered to Suzhou Jinweizhi Biotechnology Co., Ltd. synthesis. The synthetic sequence was inserted into the pET28a vector through the restriction site mentioned above to construct pET28-dypB.

[0053] Using E.coli as a template, using primer 5 with the nucleotide sequence of Seq ID No.5 and primer 6 with the nucleotide sequence of Seq ID No.6, PCR amplifies the cspA gene promoter, and adds enzyme cutting sites at both ends Point XbaI and NcoI and insert this restriction site into pET28-dypB to construct pCspA-dypB.

Embodiment 3

[0054] Example 3 Expression and purification of dypB protein

[0055] The plasmid pCspA-dypB was introduced into BL21-AL and BL21(DE3) expression hosts respectively, and positive single colonies were cultured at 37°C until OD 600 When = 0.8, add IPTG to a final concentration of 0.5mM, and induce at 16°C for 20 hours; collect the bacteria and resuspend them with 1 / 4 of the bacterial volume in buffer (20mM Tris-HCl, 5% glycerol), sonicate, and collect Supernatant; purified by nickel affinity chromatography: 20mM Tris-HCl, 500mM NaCl, 5mM imidazole equilibrated, 20mM Tris-HCl, 500mM NaCl, 60mM imidazole eluted impurity protein, 20mM Tris-HCl, 500mM NaCl, 500mM imidazole eluted target protein. The eluate was replaced with 20mM Tris-HCl, 5% glycerol dialyzed for 12 hours, protein quantification was performed with BCA protein quantification kit, and the concentration was adjusted to 1 mg / ml and stored at -80.

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Abstract

The invention belongs to the technical field of gene engineering and particularly relates to a DypB active protein heterology high expression method and application. Aiming at the problem that existing DypB protein has no activity in hyterology expression and only can generate activity by being combined with heme in vitro, the invention provides the DypB active protein heterology high expression method and application. The method disclosed by the invention comprises the steps of integrating related genes expressing the heme into host cells through genetic recombination to enable a host to have high-concentration heme, then constructing DypB into a recombinant vector and leading the recombinant vector into an expression host with high heme expression; thus, heterology high expression of the DypB active protein can be achieved. According to the method disclosed by the invention, the heme is integrated in the expression host to be synthesized into the related genes to improve the in-vivo accumulation concentration of the heme, then the DypB is expressed a lot through a cold induction mode, and the DypB has activity for degrading lignin after being expressed by the host.

Description

technical field [0001] The invention belongs to the technical field of engineering, and in particular relates to a method and application for heterologous high expression of DypB active protein. Background technique [0002] Lignin is an amorphous, aromatic polymer containing oxyphenylpropanol or its derivatives in its molecular structure widely present in plants. Lignin is a natural aromatic polymer, which is a complex, amorphous three-dimensional space structure formed by the polymerization of phenylpropane units through ether bonds (C-OC) and carbon-carbon bonds (C-C). Different side chain substituents of lignin can be divided into coniferyl alcohol (Coniferyl alcohol), coumarin (p-Coumarylalcoho1) and sinapyl alcohol (Sinapyl alcohol) and other structural units. These structural units are diverse, and the repeating units lack regularity and order. [0003] In nature, lignin is almost all associated with cellulose. Lignin and hemicellulose are covalently bonded to embed...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/08C12N15/70C12N1/21C12R1/19
CPCC12N9/0065C12N15/70C12N2800/101C12Y111/01019
Inventor 黄非田润宋乐元李谦杨明辉屈刚
Owner 四川爱奇生物科技有限公司
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