Method for producing bile salt hydrolase by fermentation of twin-arginine signal peptide and application thereof

A bile salt hydrolase, double arginine technology, applied in the directions of hydrolase, biochemical equipment and methods, enzymes, etc., can solve the problems of reducing the expression amount of target protein in fermentation supernatant, and folding can not be carried out effectively.

Inactive Publication Date: 2014-04-30
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, post-translational folding within the cell wall usually cannot be performed efficiently for some heterologous proteins that are degraded by extracellular proteases after secr...

Method used

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  • Method for producing bile salt hydrolase by fermentation of twin-arginine signal peptide and application thereof
  • Method for producing bile salt hydrolase by fermentation of twin-arginine signal peptide and application thereof
  • Method for producing bile salt hydrolase by fermentation of twin-arginine signal peptide and application thereof

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Experimental program
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Embodiment 1

[0074] Embodiment 1: the construction of recombinant bacteria

[0075] 1) The construction process of the recombinant plasmid is as follows: figure 1 as shown, figure 1 : (a) Construction process of expression plasmid pET-20b(+)-SP(Tat)-bsh, SP(Tat), signal peptide of Tat pathway; (b) Schematic diagram of fusion protein SP(Tat)-BSH. The signal peptide of the double-arginine pathway is directly fused to BSH. The underlined part is the double arginine motif "SRRXXXX"; the arrow indicates the cleavage site of signal peptidase I.

[0076] The specific operation is as follows: using the plasmid pUC57-Tat as a template, using primers torA (F) and torA (R) to amplify the torA gene encoding the TorA signal peptide by PCR ( figure 2 a), PCR conditions are: 95°C pre-denaturation for 10 minutes; 98°C for 10s, 55°C for 30s, 72°C for 20s, 30 cycles; 72°C for 10 minutes. At the same time, using the genomic DNA of L. plantarum BBE7 as a template, using torA-bsh(F) and bsh(R) as primers,...

Embodiment 2

[0079] Example 2: Induced expression of recombinant bacterial BSH and assay of enzyme activity and protein electrophoresis

[0080] The seeds cultivated overnight at 20°C and 200rpm were transferred to 25mL TB medium with a 2% inoculum, and cultivated to OD at 37°C and 200rpm 600 =0.6, add 0.4mM IPTG to induce 6h; through SDS-PAGE and enzyme activity assay, analyze the expression of bile salt hydrolysis enzyme.

[0081] The effects of three signal peptides on the intracellular expression of BSH were analyzed by SDS-PAGE. The expression of BSH in the recombinant bacterial cell is as follows image 3 a, 3c, 3e and 3g. image 3 (a), (c), (e), (g) are the whole cell protein; (b), (d), (f), (h) are the protein in the fermentation supernatant; M, protein molecular weight standard ; -, not induced with IPTG; +, induced with 0.4 mM IPTG. It can be seen from the figure that BSH is expressed to varying degrees in all recombinant bacteria. However, the molecular weight of these reco...

Embodiment 3

[0091] Embodiment 3: Optimization of BSH activity outside the recombinant bacteria

[0092] In E. coli, the composition of the medium and the fermentation conditions, such as temperature, concentration of inducer, and other parameters, will affect the secretion and expression of heterologous proteins. This chapter uses an orthogonal experimental design (L9(3 4 )) studied the induction temperature, inoculum size, induction OD 600 And the effect of IPTG concentration on the extracellular BSH activity of recombinant E.coli BL21(DE3)pLysS(pET-20b(+)-dmsA-bsh). The experiment was designed and analyzed by the software Orthogonal Design Assistant 3.1.1. The specific experimental design and results are shown in Table 2. These fermentation conditions have a great impact on the activity of recombinant bacterial extracellular BSH, and their influence order is: induction temperature > IPTG concentration > induction OD 600 > Inoculum volume. When induction temperature, IPTG concentrat...

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Abstract

The invention discloses a method for producing bile salt hydrolase by fermentation of twin-arginine signal peptides and application thereof, and belongs to the technical field of gene engineering. According to the method provided by the invention, a twin-arginine signal peptide gene and a bile salt hydrolase gene are fused, the fusion gene is transformed into the E.coli host to obtain recombinant bacteria, and recombinant bacteria is used for fermentation for producing bile salt hydrolase. The bile salt hydrolase is successfully secreted to the outside of the cells of the recombinant bacteria, and the extracellular BSH activity of the recombinant E.coli BL21 (DE3) pLysS (pET-20b (+)-dmsA-bsh) reaches the highest value of 0.72 +/- 0.05 U/mL. The invention lays foundation for the study on isolation and purification and enzymatic characteristics of BSH, and provides effective methods for secretion expression of other heterologous protein.

Description

technical field [0001] The invention relates to a method and application for promoting gene expression of bile salt hydrolase by using a double arginine signal peptide, and belongs to the technical field of genetic engineering. Background technique [0002] Bile salt hydrolase (BSH) is a metabolite of lactic acid bacteria. In addition to hydrolyzing bound bile salts, BSH can also improve the survival rate of the bacteria, promote the nutritional value of the bacteria, change the characteristics of the cell membrane of the bacteria, and remove the toxic effect of bile on the bacteria; for the host, BSH can reduce serum cholesterol , Weaken the body's digestion and absorption of fat. The bacteria with BSH enzyme activity that have been detected so far include Lactobacillus, Bifidobacterium, Enterococcus, Clostridium, Bacteroides and Streptococcus. However, there are few domestic patents and articles on the use of Escherichia coli to produce bile salt hydrolase. [0003] Sin...

Claims

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Application Information

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IPC IPC(8): C12N9/80C12N15/70
CPCC12N9/80C12N15/70C12Y305/01024
Inventor 陈坚张娟董自星周晓玲堵国成李华钟
Owner JIANGNAN UNIV
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