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Application of EDTA (ethylene diamine tetraacetic acid) in improving exocytosis volume and expression volume of escherichia coli recombinant protein

A technology of Escherichia coli and extracellular secretion, which is applied in the biological field and can solve the problems of low expression of exogenous recombinant proteins, inactive inclusion bodies, and inability to secrete

Inactive Publication Date: 2013-06-12
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Escherichia coli has seriously hindered its industrial production application due to the permeability barrier formed by its special cell wall structure. The main limiting factors are: (1) the foreign recombinant protein can only be expressed in the cell and cannot be secreted outside the cell; 2) In the process of intracellular expression of foreign recombinant protein, it is easy to form inactive inclusion bodies, which seriously reduces the activity quality of recombinant protein; (3) The expression level of foreign recombinant protein is generally low

Method used

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  • Application of EDTA (ethylene diamine tetraacetic acid) in improving exocytosis volume and expression volume of escherichia coli recombinant protein
  • Application of EDTA (ethylene diamine tetraacetic acid) in improving exocytosis volume and expression volume of escherichia coli recombinant protein
  • Application of EDTA (ethylene diamine tetraacetic acid) in improving exocytosis volume and expression volume of escherichia coli recombinant protein

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Embodiment 1

[0047] (1) Materials

[0048] 1. Strains and plasmids

[0049] Bacillus sp.III-3 (preservation number: CGMCC No.2138, preservation date: August 22, 2007, patent number: 200710030787.6, name: an alkalophilic bacillus and the endoglucan produced by it Carbohydrase and Application) is used as the original bacteria of the alkaline cellulase gene, E.coli Top10 (Invitrogen) is used for the maintenance and operation of the plasmid, and E.coli BL21Star (DE3) (Invitrogen) is used as the expression host bacteria. The expression plasmid is pET-28a (+) (Invitrogen company, its plasmid map is as follows figure 1 As shown, the map of multiple cloning sites is shown in figure 2 shown).

[0050] 2. Medium

[0051] Each liter medium contains: 10g compound peptone, 5g yeast powder, 5NaCl, pH value is 7.

[0052] 3. Instrument

[0053] MyCyclerTM thermal cycler PCR instrument, DNA and protein electrophoresis system are products of Bio-rad; spectrophotometer and micro-spectrophotometer are p...

Embodiment 2

[0105] (1) Experimental method

[0106] 1. Construction of recombinant Escherichia coli

[0107] Bacillus subtills.32356 (purchased from China Industrial Microorganism Culture Collection Management Center, preservation number: CICC No.20636, as the original bacterium of amylase gene) genome acquisition method refers to the instructions of Omega gene extraction kit; DNA restriction enzyme Cutting, ligation, and PCR amplification all refer to the instructions of Takera reagents; the primers (Shanghai Sangong Synthetic) used to amplify the amylase gene are:

[0108] Upstream primer (Nde I): 5′-GTC CATATG GTAAATGGCACGCTGATG-3′;

[0109] Downstream primer (BamH I): 5′-CGC GGATCC TTATTTCTGAACATAAATGGAGAC-3';

[0110] The PCR product and the expression plasmid pET-28a(+) were ligated and transformed into E.coli Top10 after double enzyme digestion to obtain the recombinant plasmid pET-28a-A, transformed into E.coli BL21Star(DE3), and subjected to liquid PCR and sequencing (nucle...

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Abstract

The invention discloses application of EDTA (ethylene diamine tetraacetic acid) in improving exocytosis volume and expression volume of escherichia coli recombinant protein. The inventor of the invention discovers that EDTA has performance of improving exocytosis volume and expression volume of escherichia coli recombinant protein, and has the best effect in improving the exocytosis volume and expression volume of escherichia coli recombinant protein as compared with EGTA (ethylene glycol tetraacetic acid), citrulline, guanidine hydrochloride and Triton X-100. In the application method disclosed by the invention, after an inducer performs inducing culture to escherichia coli containing recombinant vector, EDTA or EDTA and lysozyme are added to continue culturing. The application method ofthe EDTA in improving the exocytosis volume and expression volume of the escherichia coli recombinant protein, disclosed by the invention, has simple operation and low cost, and the exocytosis volumeand expression volume of protein with larger molecular weight and difficult excretion can be increased.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an application of EDTA in improving the extracellular secretion and expression of Escherichia coli recombinant proteins. Background technique [0002] Escherichia coli expression system is the earliest and most widely used expression system in laboratory applications and even in industrial production. Its natural characteristics endow it with great application value. These characteristics include: (1) Its whole genome sequencing is completed, and its genetic background is very detailed; (2) The gene cloning and expression system is mature and perfect; (3) It reproduces rapidly, is easy to cultivate, and operates Convenience and genetic stability; (4) The cells themselves express fewer protein components and low expression levels, and are easy to purify and recover foreign recombinant proteins; (5) Approved by the US FDA as a safe genetic engineering recipient organism. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N9/26C12R1/19
Inventor 邢苗刘森林陈伟钊杜坤
Owner SHENZHEN UNIV
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