A method for promoting extracellular expression of target protein and its application

A technology of target protein and protein, which is applied in the field of promoting the extracellular expression of target protein, can solve problems such as unfavorable fermentation regulation and industrial amplification, and achieves the improvement of the ratio of extracellular enzyme activity and production intensity, the improvement of production intensity, and the convenient regulation of enzyme production process. Effect

Active Publication Date: 2018-08-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, one of the products of T.fusca cutinase hydrolyzing phospholipid molecules is lysophospholipid, which has the function similar to surfactant and will induce a large amount of foam during fermentation, which is not conducive to fermentation regulation and industrial scale-up

Method used

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  • A method for promoting extracellular expression of target protein and its application
  • A method for promoting extracellular expression of target protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Expression of Phospholipase C in E. coli BL21 (DE3)

[0025] Construction of pETDuet-1-plc recombinant plasmid: pETDuet-1 plasmid (purchased from Novagen) and pMD18-T-plc carrying phospholipase C gene (amino acid sequence shown in SEQ ID NO.1) were subjected to Nco I, After double digestion with Hind Ⅲ, the digestion product was recovered by gel tapping, T4 ligase was ligated overnight at 16 °C, and the ligation product was transformed into E. coli JM109, which was coated on 100 μg·mL ampicillin -1 The LB agar plates were cultured at 37 °C for 8-10 h, and the transformants were selected with an ampicillin content of 100 μg·mL. -1 The LB liquid medium was cultured for 8-10h, and the plasmid was extracted to obtain the recombinant plasmid pETDuet-1-plc.

[0026] Expression and preparation of phospholipase C: The pETDuet-1-plc plasmid was transformed into E.coli BL21 (DE3) host bacteria, and the ampicillin content was 100 μg·mL -1 Cultured on LB agar plates at...

Embodiment 2

[0028] Example 2: Co-expression of xylanase and phospholipase C in E. coli 3L tank fermentation

[0029] Construction of pETDuet-1-plc-XynA recombinant plasmid: PETDuet-1-plc plasmid and pET20b(+)-XynA of the xylanase gene with NCBI number AGM16410 cloned into pET20b(+) were subjected to Nde I and Xho I Double-enzyme digestion, the digestion product was recovered by gel cutting, and then ligated with T4 ligase at 16°C overnight. The ligation product was transformed into E.coli JM109 competent cells, and the transformation product was coated with ampicillin with a content of 100 μg·mL -1 The LB agar plates were cultured at 37 °C for 8-10 h, and the transformants were selected with an ampicillin content of 100 μg·mL. -1 The LB liquid medium was cultured for 8-10h, and then the plasmid was extracted to obtain the recombinant plasmid pETDuet-1-plc-XynA.

[0030] Expression and preparation of xylanase: The plasmid pETDuet-1-plc-XynA was transformed into E.coli BL21(DE3) host bacte...

Embodiment 3

[0034] Example 3: Co-expression of Glucose Isomerase and Phospholipase C in E. coli 3L Tank Fermentation

[0035] Construction of pETDuet-1-plc-glu recombinant plasmid: pETDuet-1-plc plasmid and pET24a(+)-glu of the glucose isomerase gene with NCBI number AAZ55638 cloned into pET24a(+) were subjected to Nde I and Xho I double-enzyme digestion, the digestion product was recovered by gel cutting, and then ligated with T4 ligase at 16°C overnight. The ligation product was transformed into E.coli JM109 competent cells, and the transformation product was coated with ampicillin content of 100 μg·mL -1 The LB agar plates were cultured at 37 °C overnight, and the transformants were selected with an ampicillin content of 100 μg·mL. -1 The LB liquid medium was cultured, and then the plasmid was extracted to obtain the recombinant plasmid pETDuet-1-plc-glu.

[0036] Expression and preparation of glucose isomerase: pETDuet-1-plc-glu was transformed into E.coli BL21(DE3) host bacteria, an...

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Abstract

The invention discloses a method for promoting extracellular expression of target protein and an application of the method, and belongs to the technical field of bioengineering. Bacillus cereus phospholipase C and the target protein (including intracellular location protein and extracellular location protein) are subjected to coexpression in escherichia coli, so that the production intensity of the extracellular location target protein can be improved; extracellular expression of the intracellular location target protein is realized; the production efficiency is improved; the post-extraction technology is simplified; and the method has relatively high academic significance and application value.

Description

technical field [0001] The invention relates to a method for promoting the extracellular expression of a target protein and its application, and belongs to the technical field of bioengineering. Background technique [0002] There are two localization methods of recombinant proteins expressed by genetically engineered bacteria: intracellular localization, with the help of a series of chaperone proteins, the protein synthesized in the ribosome is localized in the cell matrix; extracellular localization, after synthesis in the ribosome, The target protein is transported to the extracellular matrix under the action of its own functional sequence and the secretion system of the host bacteria. Among these two positioning methods, extracellular positioning not only helps protein folding, but also helps to reduce the amount of inclusion bodies formed. At the same time, it can avoid operations such as breaking cells during downstream purification, simplify purification steps, reduce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/67C12N15/70C12N9/92C12N9/42C12R1/19
Inventor 吴敬宿玲恰姜琪
Owner JIANGNAN UNIV
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