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Method for efficient expression and secretion of transpeptidase Sortase A

A technology of transpeptidase and expression vector, which is applied in the field of genetic engineering, can solve the problems of the extracellular secretion of the added concentration and the added time of the target protein, inconsistent effects, inconsistent effects, etc., to shorten the fermentation time, save production costs, and realize industrial production Effect

Active Publication Date: 2015-12-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effects of different chemical reagents are inconsistent, and the effects of the same chemical reagent on different target proteins or different hosts are also inconsistent. The concentration and time of adding the same chemical reagent will also have a significant impact on the extracellular secretion of the target protein.

Method used

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  • Method for efficient expression and secretion of transpeptidase Sortase A
  • Method for efficient expression and secretion of transpeptidase Sortase A
  • Method for efficient expression and secretion of transpeptidase Sortase A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Extracellular production of SortaseA with Escherichia coli E.coliBL21 (DE3)

[0026] The construction method of the recombinant Escherichia coli used in the present invention to produce Sortase A is: take the genome of S.aureus as a template, amplify with srt-F and srt-R (Table 1) primers to remove the N-terminal signal peptide (59 amino acids) The srt gene and the T vector were ligated and transformed into E.coliJM109. After the correctness was verified by sequencing, they were subcloned into plasmids pET22b and pET20b and transformed into E. coli expression host E.coliBL21(DE3). The recombinant Escherichia coli (pET22b-srt and pET20b-srt) were induced and expressed in 250mL containing 25mL medium, the induction temperature was 30°C, the rotation speed was 250rpm, and the final concentrations of the inducer IPTG were 1mM / L and 0.4mM respectively / L. Fermentation results showed that active Sortase A could be detected in the fermentation broth of the strain us...

Embodiment 2

[0027] The comparative screening of embodiment 2 different additives

[0028]In order to evaluate the effect of different additives on the extracellular production of SortaseA in Escherichia coli, the effect of different additives on the extracellular SortaseA enzyme activity was screened first. Additives were added together with IPTG during induction. Or the cell wall all has a destructive effect, considering the cost and toxicity of the additive, SDS, glycine, Tween 80, Triton X-100 are selected as the additive of the preliminary test, the result ( figure 1 ) shows that adding 0.5% (m / v) glycine during induction has obvious promotion effect on extracellular enzyme activity, and adding 5% Tween 80 or 0.5% Triton 100 promotes the extracellular enzyme activity of SortaseA The effect is almost 0. Therefore, glycine was selected as the additive for the next experiment, and other additives were not studied here.

Embodiment 3

[0029] The screening of embodiment 3 different concentrations of glycine

[0030] In order to compare the influence of adding different final concentrations (m / v) glycine on E. coli extracellular production of SortaseA when inducing, selected 0.5%, 0.75%, 1.0%, 1.5%, 2.0%, 2.5% six kinds of addition levels, the result ( figure 2 ) shows that 0.75% and 1.0% are more obvious to the extracellular production promotion effect of SortaseA, but considering additive cost and thalline growth ( image 3 ), the added concentration of 0.75% is preferred.

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Abstract

The invention discloses a method for efficient expression and secretion of transpeptidase Sortase A, and belongs to the field of genetic engineering. The method disclosed by the invention successfully achieves the efficient extracellular production of transpeptidase Sortase A in recombinant escherichia coli BL21 (DE3) by virtue of an addition strategy of adding glycine during inducing, so as to solve the existing problems of low transpeptidase Sortase A secretion rate and long fermentation cycle. By optimizing a target protein carrier as well as an additive and an addition strategy in a fermentation system, the method disclosed by the invention discovers that pET-22b carrier is suitable for the extracellular production of the Sortase A; and the method can be used for greatly improving the Sortase A extracellular production level of the escherichia coli by adding glycine in two stages (first stage: adding glycine which is 0.5% in final concentration during inducing, and second stage: adding 2% of glycine after 6h of inducing). The method disclosed by the invention achieves the efficient extracellular production of transpeptidase Sortase A in escherichia coli for the first time, so as to lay a foundation for subsequent Sortase A industrial production.

Description

technical field [0001] The invention relates to a method for efficiently secreting and expressing transpeptidase SortaseA, which belongs to the field of genetic engineering. Background technique [0002] Sortase A, found in Gram-positive bacteria, is a transpeptidase that mediates the covalent attachment of cell wall-anchored proteins to the cell wall. SortaseA not only has high catalytic reaction efficiency, but also its target protein must have a specific recognition sequence LPXTG (X is any amino acid). Since the discovery of SortaseA, its scope of application has continued to expand, involving fields such as vaccine development, nucleic acid modification, protein modification, protein-peptide linkage and immobilization. [0003] In 2001, Schneewind's research group successfully expressed SortaseA in E.coliBL21(DE3) using pET9a vector for the first time. Its protein expression can meet the needs of laboratory-level research on SortaseA, but it is not conducive to the pu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/10C12R1/19
Inventor 吴志猛赵鑫锐洪皓飞
Owner JIANGNAN UNIV
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