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Method for efficiently producing transpeptidase sortase A by using recombinant escherichia coli

A technology of recombinant Escherichia coli and transpeptidase, applied in the field of genetic engineering, can solve problems such as the influence of protein structure, and achieve the effect of efficient production

Inactive Publication Date: 2016-06-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the tags on these two vectors are fused with SortaseA, the solubility and expression of SortaseA in E. coli cells can be enhanced, but this method may affect the overall protein structure of SortaseA

Method used

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  • Method for efficiently producing transpeptidase sortase A by using recombinant escherichia coli
  • Method for efficiently producing transpeptidase sortase A by using recombinant escherichia coli
  • Method for efficiently producing transpeptidase sortase A by using recombinant escherichia coli

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1 Recombinant E. coli fermentation produces SortaseA

[0024] Using the genome of S.aureus as a template, amplified with srt-F and srt-R (Table 1) primers to obtain the srt gene that removes the N-terminal signal peptide, and transforming it into E.coliJM109 by connecting with the T vector. Then it was subcloned into plasmid pET28a and transformed into E. coli expression host E.coliBL21(DE3).

[0025] With TB medium (peptone 12g / L, yeast powder 24g / L, glycerol 4g / L, KH 2 PO 4 23.1g / L, K 2 HPO 4 125.4g / L) as fermentation medium, 50mL of 500mL shake flask liquid, 2% inoculum, fermentation temperature 37°C, induction fermentation time 3h, inducer IPTG concentration 100mM / L, induction timing OD 600 = 0.6. The yield of SortaseA was 135.4mg / L, and the enzyme activity per unit mass of bacteria was 692.1U / mg.

Embodiment 2

[0026] Embodiment 2 produces SortaseA with different substratum fermentation

[0027]Different carbon sources (dextrin, soluble starch, lactose, galactose, sucrose, fructose, glucose, maltose, glycerin), nitrogen sources (peptone, industrial grade tryptone, industrial grade beef extract, casein, citric acid Triammonium, ammonium chloride, ammonium sulfate, fish powder peptone, beef extract, soybean peptone, urea) replace the corresponding components in the TB medium or add different additives (yeast powder, industrial grade yeast powder, phosphate buffer, MOPS) and metal ions (CaCl 2 、CuSO 4 , FeSO 4 、CoCl 2 , MnSO 4 , LiCl, ZnCl 2 , MgSO 4 , NaCl, NiSO 4 ). In the carbon source and nitrogen source tests, the highest value in the measured enzyme activity is defined as 100%, and the rest of the enzyme activities are relative enzyme activities compared with the highest enzyme activity; The enzyme activity measured during the addition (control) was defined as 100%, and t...

Embodiment 3

[0028] Embodiment 3 Fermentation produces SortaseA with different fermentation conditions

[0029] Fermentation and production of Sortase A with different fermentation temperatures, fermentation times and medium initial pH, Figure 5 , 6 The results showed that when the recombinant Escherichia coli was induced at 16°C, although the growth of the bacteria was slow, the activity of Sortase A was the highest after 4 hours of fermentation; followed by the induction at 20°C for 8 hours, but there was also the problem of slow growth of the bacteria; and then the induction at 30°C 4h, the fermentation temperature is suitable for Escherichia coli growth, and the obtained enzyme activity is also higher ( Figure 5 ). Although the enzyme activity induced by low temperature is the highest, it is not suitable for industrial production due to the defects of slow growth of bacteria and high cost of controlling low temperature. Therefore, the optimum fermentation temperature for enzyme pro...

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Abstract

The invention discloses a method for efficiently producing transpeptidase sortase A by using recombinant escherichia coli, and belongs to the field of gene engineering. Efficient production of the sortase A in the escherichia coli is successfully achieved, the problem of lower expression level of the enzyme reported at present is solved, production is performed in a 7 L fermentation tank, and the yield of the sortase A can reach 295.2 mg / L and is increased by 58.5% than the yield before optimization; the enzyme activity of unit mass of thalli can reach 4845 U / mg and is increased by 52.8% than the activity before optimization. By system optimization of recombinant strain fermentation production conditions, a foundation is laid for subsequent sortase A industrialized production.

Description

technical field [0001] The invention relates to a method for efficiently producing transpeptidase Sortase A by using recombinant Escherichia coli, belonging to the field of genetic engineering. Background technique [0002] SortaseA is a transpeptidase that mediates the covalent binding of cell wall-anchored proteins to the cell wall in Gram-positive bacteria. Because SortaseA has both protease and transpeptidase activities, its application in the field of biotechnology has attracted more and more attention in recent years. Since the first report of using SortaseA as a protein or polypeptide linking tool in 2004, the linking reaction involving SortaseA has rapidly become a research hotspot and has been widely used in biochemistry, proteomics, biomedicine, and bioengineering technology. The SortaseA-mediated protein ligation method can overcome the complex preparation process, damage to protein activity, and heterogeneous ligation products of the traditional chemical ligatio...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N15/70C12N1/21C12R1/19
CPCC12N9/52C12Y304/2207
Inventor 吴志猛赵鑫锐洪皓飞王珣
Owner JIANGNAN UNIV
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