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A method for efficiently secreting and expressing transpeptidase sortase A

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

Active Publication Date: 2018-04-06
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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  • A method for efficiently secreting and expressing transpeptidase sortase A
  • A method for efficiently secreting and expressing transpeptidase sortase A
  • A method for efficiently secreting and expressing transpeptidase sortase A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Extracellular production of Sortase A by E. coli BL21 (DE3)

[0026] The construction method of the recombinant Escherichia coli for producing Sortase A used in the present invention is as follows: taking the genome of S. aureus as a template, and amplifying the N-terminal signal peptide (59 amino acids) with srt-F and srt-R (Table 1) primers The srt gene was ligated with the T vector and transformed into E.coli JM109. After the sequencing was verified to be correct, it was subcloned into plasmids pET22b and pET20b and transformed into E.coli expression host E.coli BL21 (DE3) respectively. The recombinant Escherichia coli (pET22b-srt and pET20b-srt) were induced to express in 250 mL of 25 mL of medium, the induction temperature was 30 °C, the rotational speed was 250 rpm, and the final concentrations of the inducer IPTG were 1 mM / L and 0.4 mM, respectively. / L. The fermentation results showed that the active Sortase A could be detected in the fermentation bro...

Embodiment 2

[0027] Comparative Screening of Different Additives in Example 2

[0028]In order to evaluate the effects of different additives on the extracellular production of Sortase A in Escherichia coli, the effects of different additives on the extracellular Sortase A activity were first screened. The additives were added together with IPTG during induction. Bacillus cell membrane or cell wall has a damaging effect. Considering the cost and toxicity of additives, SDS, glycine, Tween 80, and Triton X-100 were selected as additives in the preliminary test. The results ( figure 1 ) showed that the addition of 0.5% (m / v) glycine significantly promoted the extracellular enzyme activity during induction, while the addition of 5% Tween 80 or 0.5% triton 100 had a significant effect on the extracellular enzyme activity of Sortase A The facilitation effect is almost 0. Therefore, glycine was selected as the additive in the next experiment, and other additives will not be studied here.

Embodiment 3

[0029] Example 3 Screening of different concentrations of glycine

[0030] In order to compare the effects of adding different final concentrations (m / v) of glycine on the extracellular production of Sortase A in E. coli during induction, six addition levels of 0.5%, 0.75%, 1.0%, 1.5%, 2.0% and 2.5% were selected. result( figure 2 ) showed that 0.75% and 1.0% had more obvious promotion effects on the extracellular production of Sortase A, but considering the cost of additives and the growth of bacteria ( image 3 ), the addition 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 a transpeptidase Sortase A, belonging to the field of genetic engineering. Background technique [0002] Sortase A is found in Gram-positive bacteria and is a transpeptidase that mediates the covalent binding of cell wall anchoring proteins to the cell wall. Sortase A not only catalyzes the reaction with high efficiency, but also the target protein it acts on must have a specific recognition sequence LPXTG (X is any amino acid). Since the discovery of Sortase A, its application range has been continuously expanded, involving fields such as vaccine development, nucleic acid modification, protein modification, protein-peptide linkage and immobilization. [0003] In 2001, the Schneewind research group successfully expressed Sortase A in E.coli BL21(DE3) using pET9a vector for the first time. Its protein expression can meet the needs of the laboratory-level study of Sortase A, but...

Claims

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Application Information

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