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Method for obtaining streptomycete trypsin zymogen by utilizing recombinant gene engineering bacterium

A technology of genetically engineered bacteria and trypsin, applied in the field of genetically engineered bacteria and construction of high-yielding trypsin zymogen, to achieve the effects of high product purity, simple process, and high yield

Inactive Publication Date: 2013-01-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Streptomyces trypsin (SGT) (E.C.3.4.21.4) is an alkaline serine protease derived from Streptomyces griseus. Sequencing found that in the gene SprT encoding SGT, the SGT signal peptide and streptomyces active trypsin were removed, and there was also a leader peptide (-Ala-Pro-Asn-Pro-) at the N-terminus of the mature enzyme (-Ala-Pro-Asn-Pro-) ( figure 1 ); Through previous studies on the secretion, purification, amino acid sequencing and related physiological and biochemical functions of Streptomyces trypsin in Streptomyces griseus, it was found that when Streptomyces aerial hyphae and vegetative hyphae differentiate, Streptomyces extracellular Active trypsin appears, and purification and amino acid sequencing can only obtain the information of active trypsin, but the activation and activation mechanism of Streptomyces trypsinogen with leader peptide has not been reported, and for Streptomyces trypsin The acquisition of the zymogen has not been reported, so the establishment of a method for obtaining the trypsin zymogen with a wild leader peptide at the N-terminus is crucial for the study of the crystal structure of the Streptomyces trypsin zymogen and the study of its activation mechanism significance

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  • Method for obtaining streptomycete trypsin zymogen by utilizing recombinant gene engineering bacterium
  • Method for obtaining streptomycete trypsin zymogen by utilizing recombinant gene engineering bacterium
  • Method for obtaining streptomycete trypsin zymogen by utilizing recombinant gene engineering bacterium

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Embodiment 1

[0037] Embodiment 1: the acquisition of trypsin zymogen gene

[0038] The trypsinogen gene is derived from the coding sequence of the trypsinogen gene in Streptomyces griseus (GenBank Accession No. M64471), in which the 628th to 1307th are the genes encoding pmt. After analyzing the codon bias of the gene, EcoRI and NotI restriction sites were added to its 5' end and 3' end respectively. The nucleotide sequence of the SprT gene is: SEQ ID NO.1, using chemical methods Perform total synthesis.

Embodiment 2

[0039] Example 2: Construction of recombinant plasmid pPIC9K-pmt containing trypsin zymogen gene (pmt)

[0040] The in vitro amplified fragment of the pmt gene and the expression vector pPIC9K were respectively digested with NotI and EcoRI, and ligated with T4 ligase after recovery. The ligation reaction system is (10 μL): target gene fragment 2 μL, carrier DNA 2 μL, 10×T4 ligase Buffer 1 μL, T4 DNA ligase 1 μL, ddH 2 04 μL.

[0041] The ligation product was transformed into competent Escherichia coli JM109 for transformation. The conversion method is as follows:

[0042] (1) Under sterile conditions, take 200 μL of competent cells and place them in a sterile microcentrifuge tube;

[0043] (2) Add 1-2 μL of recombinant plasmid to each tube, rotate gently to mix the contents, and place on ice for 30 minutes;

[0044] (3) Heat shock at 42°C for 90sec (accurate), do not shake the centrifuge tube;

[0045] (4) Quickly transfer the centrifuge tube to an ice bath to cool the ce...

Embodiment 3

[0049] Embodiment 3: the construction of trypsin zymogen genetically engineered bacteria

[0050] The expression vector pPIC9K-pmt was linearized by cutting with SalI. Enzyme digestion system (50 μL system): recombinant plasmid 10 μL, Buffer 5 μL, SalI 3 μL, ddH 2 O32 μL. Water bath at 37°C for 3 hours, purify and recover the linearized product with a PCR product purification kit, and transform Pichia pastoris GS115 by electric shock method, the specific method is as follows:

[0051] (1) Inoculate a loop of activated Pichia pastoris GS115 in 25mL of LYPD liquid medium, and culture overnight at 28°C to 30°C with shaking;

[0052] (2) Transfer the above culture solution into 100mL YPD (500mL Erlenmeyer flask) liquid culture medium, shake culture at 28℃~30℃, measure every 1h, and cultivate until the cell concentration OD 600 1.3~1.5;

[0053] (3) Cool in ice water for more than 10 minutes;

[0054] (4) Collect the bacteria by centrifugation at 4°C and 8000r / min, suspend the...

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Abstract

The invention discloses a method for obtaining streptomycete trypsin zymogen by utilizing a recombinant gene engineering bacterium. By connecting chemical fully-synthetic trypsin zymogen onto a Pichia pastoris GS115 chromosome, a gene engineering bacterium capable of achieving efficient secretory expression of the trypsin zymogen is constructed. After the gene engineering bacterium is fermented for 3-4 days, the expression of the streptomycete trypsin zymogen is obtained, and the problem that the trypsin zymogen can not be obtained from wild streptomyces griseus for a long time is solved. The trypsin zymogen produced by the gene engineering bacterium is high in yield, simple in process and favorable for performing fundamental researches on streptomycete crystal structures and the activation mechanism of the streptomycete crystal structures.

Description

technical field [0001] The invention relates to a method and application for obtaining streptomyces trypsinogen by using recombinant genetically engineered bacteria, in particular to a genetically engineered bacteria with high yield of trypsinogen and its construction method and application, belonging to the field of biotechnology. Background technique [0002] Streptomyces trypsin (SGT) (E.C.3.4.21.4) is an alkaline serine protease derived from Streptomyces griseus. Sequencing found that in the gene SprT encoding SGT, the SGT signal peptide and streptomyces active trypsin were removed, and there was also a leader peptide (-Ala-Pro-Asn-Pro-) at the N-terminus of the mature enzyme (-Ala-Pro-Asn-Pro-) ( figure 1 ); Through previous studies on the secretion, purification, amino acid sequencing and related physiological and biochemical functions of Streptomyces trypsin in Streptomyces griseus, it was found that when Streptomyces aerial hyphae and vegetative hyphae differentiate,...

Claims

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Application Information

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IPC IPC(8): C12N9/76C12N15/57C12N15/81C12N1/19C12R1/84
Inventor 陈坚令桢明堵国成康振李江华
Owner JIANGNAN UNIV