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A method for constructing high-efficiency secretion and expression of transpeptidase sortase A in Escherichia coli

A technique for secreting, expressing, and recombining Escherichia coli, which is applied in the field of genetic engineering and can solve the problems of time-consuming, cost, and low yield

Active Publication Date: 2020-07-07
JIANGNAN UNIV
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  • Application Information

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Problems solved by technology

The intracellular expression of Sortase A has the following major defects: (1) low yield: in existing reports, the highest yield is only 76.9mg / L; (2) the Sortase A expressed in the cell is easy to Inclusion bodies are formed in cells, and a time-consuming and costly cell disruption step is required prior to purification

Method used

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  • A method for constructing high-efficiency secretion and expression of transpeptidase sortase A in Escherichia coli
  • A method for constructing high-efficiency secretion and expression of transpeptidase sortase A in Escherichia coli
  • A method for constructing high-efficiency secretion and expression of transpeptidase sortase A in Escherichia coli

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Experimental program
Comparison scheme
Effect test

specific Embodiment approach

[0010] The method for the efficient secretion of Sortase A by recombinant escherichia coli is: inoculate the cultivated seeds (LB medium) into the 500mL Erlenmeyer flask with 2% inoculum size; the fermentation medium is TB medium (peptone 12.0 g / L, yeast powder 24.0g / L, glycerin 4.0g / L, KH 2 PO 4 2.31g / L, K 2 HPO 4 12.54g / L, pH 7.0); the fermentation conditions adopted are: fermentation temperature 30°C, induction culture 36h, inducer IPTG concentration 100mM / L, induction timing OD 600 = 0.6.

[0011] Sortase A purification method: After 36 hours of induction, the bacterial solution was centrifuged at 9000 rpm for 3 minutes at 4°C and the supernatant was taken, which was the crude enzyme solution. Sortase A in the crude enzyme solution was purified with His Tag-tagged protein chelating magnetic beads (Beaver Nano Technology Co., Ltd.). For specific steps, please refer to the instructions for use of the magnetic beads.

[0012] Determination of protein content: Use the B...

Embodiment 1

[0016] Embodiment 1 Realization of high-efficiency secretion and expression of Sortase A

[0017] Using the genome of Staphylococcus aureus as a template, the srtA gene (SEQ ID NO.1) obtained by removing the N-terminal signal peptide was amplified with srt-F and srt-R (Table 1) primers, and cloned into the Nco I / Bam HI site to obtain the pET22a-PelB-srtA vector. Using the Escherichia coli genome as a template, OmpA was amplified with primers OmpA-F and OmpA-R, TorA-F and TorA-R, DmsA-F and DmsA-R, FdnG-F and FdnG-R (Table 1). , TorA, DmsA and FdnG four signal peptide coding genes, and use OmpA-SrtA-F and OmpA-SrtA-R, TorA-SrtA-F and TorA-SrtA-R, DmsA-SrtA-F and DmsA-SrtA- R, FdnG-SrtA-F and FdnG-SrtA--R (Table 1) primers amplified to the srtA gene fragment that could be fused with the four signal peptide coding genes. Using fusion PCR technology, four fragments of OmpA-SrtA, TorA-SrtA, DmsA-SrtA, and FdnG-SrtA were obtained, and the PelB signal peptide on the pET22a-PelB-sr...

Embodiment 2

[0020] Example 2 Co-expression of molecular chaperones improves the secreted expression level of Sortase A

[0021] Starting from the E.coli BL21(DE3) strain carrying the pET22a-PelB-srtA vector, after preparation of competence, five different molecular chaperone co-expression plasmids (pG-KJE8, pGro7, pKJE7, pG-Tf2 and pTf16 were transformed into To the bacterial strain. After the activation of five different molecular chaperone co-expressed bacterial strains, induce expression, detect the content and enzyme activity of extracellular Sortase A. The results (table 3) show that the effect of pG-Tf2 co-expression is the best, and the secretory expression of Sortase A The amount was further increased to 90.2mg / L, and the extracellular enzyme activity level was further increased to 34.2U / mL.

[0022] Table 1 Primers used in the construction of Sortase A secreting strains mediated by different signal peptides

[0023]

[0024]

[0025] Table 2 Primers used for Sortase A codon ...

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Abstract

The invention discloses a method for constructing Escherichia coli capable of efficiently secreting and expressing transpeptidase Sortase A, and belongs to the field of gene engineering. According tothe present invention, the efficient secretion and expression of SortaseA in Escherichia coli is successfully achieved, and the problems that the transpeptidase Sortase A cannot be secreted and expressed and the expression level of the transpeptidase Sortase A in cells is low in the existing report are solved; at the shake flask level, the yield of the obtained sortedaseA can achieve 90.2 mg / L, and the extracellular enzyme activity can achieve 34.2 U / mL; and the foundation is established for the subsequent industrial production of SortaseA.

Description

technical field [0001] The invention relates to a method for constructing Escherichia coli to efficiently secrete and express transpeptidase Sortase A, belonging to the field of genetic engineering. Background technique [0002] Sortase A is a class of transpeptidases that mediate the covalent binding of Gram-positive bacterial cell wall-anchored proteins to the cell wall. Its application in the field of biotechnology has attracted more and more attention in recent years. Since the first report of using Sortase A as a linking tool for proteins or polypeptides in 2004, the linking reaction involving Sortase A has rapidly become a research hotspot and has been widely used in biochemistry, proteomics, biomedicine, and bioengineering technologies, etc. field. But so far, there are few studies on the high-efficiency expression and industrial production of Sortase A, and the expression method of the enzyme is still mainly limited to the intracellular expression of Escherichia col...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/52C12N15/57C12R1/19
Inventor 吴志猛赵鑫锐洪皓飞邓涛
Owner JIANGNAN UNIV
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