New method for xylanase secretory expression in escherichia coli

A technology of xylanase and Escherichia coli, which is applied in the field of genetic engineering to achieve the effect of maintaining high activity, less miscellaneous protein content, and being conducive to large-scale industrial application

Pending Publication Date: 2019-05-31
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] (3) So far, there are no more reports on the secretion and expression of xylanase production in E. coli. A large number of molecular chaperones in the periplasmic space of E. coli can effectively assist the correct folding of secreted proteins in the secretory pathway. Therefore, it is important to establish a The new expression system needs to consider the selection from its own nature, and try to find an effective signal peptide to help its secretion in E. coli

Method used

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  • New method for xylanase secretory expression in escherichia coli
  • New method for xylanase secretory expression in escherichia coli
  • New method for xylanase secretory expression in escherichia coli

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Experimental program
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Effect test

Embodiment 1

[0077] Embodiment 1: the construction of alkaline xylanase XynHB escherichia coli secretion expression vector:

[0078] 1) In the specific implementation of the present invention, the alkaline xylanase XynHB is derived from the mature form of Bacillus subtilis HBP8, and the 27-amino acid signal peptide at its N-terminal has been removed. (NCBI: AY954630.1).

[0079] 2) In the specific implementation of the present invention, the secretory expression strategy is a signal peptide that can promote extracellular secretion of the protein by fusing the N-terminus of xylanase mature XynHB.

[0080] 3) The signal peptides include well-characterized signal peptides OmpA, PelB, PhoA, and Lpp derived from the Sec pathway, signal peptides TorA and Fdog from the Tat pathway, and signal peptide FhuD derived from the Sec-Tat pathway. The amino acid sequence of the signal peptide is shown in the table below

[0081]

[0082] 4) According to the Escherichia coli codon preference table, th...

Embodiment 2

[0103] Secreted expression of alkaline xylanase XynHB in E. coli:

[0104] 1) The successfully constructed XynHB expression vectors were transformed into Escherichia coli BL21(DE3) strains, spread on kana-resistant plates and cultured overnight until single clones grew out.

[0105] 2) Pick a single clone from the plate and transfer it to 3ml LB (containing 50ug / ml kanamycin), and place it on a shaker at 37°C and 200rpm for overnight culture for 12-14h.

[0106] 3) Transplant into 50ml LB (containing 50ug / ml kanamycin) with 1% inoculum size, and continue culturing at 37°C until OD. When the 6 0 0 is 0.5-0.6, add 0.5mM IPTG at a final concentration and place on a shaker at 18°C ​​for 20h.

[0107] 4) The induced sample was centrifuged in a high-speed refrigerated centrifuge at 17,000 rpm and 4° C. for 15 minutes, and the supernatant was collected to obtain a crude xylan enzyme sample secreted in the culture medium.

[0108] 5) Purification and identification of xylanase:

[...

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Abstract

The invention belongs to the technical field of genetic engineering and discloses a new method for xylanase secretory expression in escherichia coli. A signal peptide is fused to the N terminal of a xylanase gene, meanwhile, a codon of an encoding histidine tag is added to the C terminal of a xylanase gene sequence, and a target gene nucleotide sequence Signal peptide-Xylnase-6*His is obtained through PCR product recovery; in a target gene cloning and expression vector pET28a, a correct recombinant expression vector is obtained through sequencing; the recombinant expression vector is convertedto a escherichia coli strain; IPTG is added into a sample, and induction culture is carried out on the sample; efficient secretory expression of xylanase is achieved. According to the method, xylanase can be directly secreted into a culture medium; compared with original escherichia coli intracellular expression or yeast secretory expression, the high activity of xylanase can be ensured, the operation is easier, more time is saved, and the cost is lower.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. In particular, it relates to a new method for secreting and expressing xylanase in Escherichia coli. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] At present, there are two main methods for preparing xylanase: one is intracellular expression in Escherichia coli, and the target gene is cloned into the commonly used expression vectors pET28a and pET26b of Escherichia coli for routine intracellular expression, which requires complicated bacterial disruption and purification methods At the same time, problems such as low activity and low purity of xylanase due to physical effects cannot be avoided, so the existing research results do not recommend the expression of xylanase in E. coli; the other is Pichia pastoris Or secreted and expressed by Saccharomyces cerevisiae. Yeast cells have the characteristics of lon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N15/66
Inventor 易犁张发英何华华喻婵
Owner HUBEI UNIV
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