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Method for optimizing signal peptide and improving exocytosis expression of trypsin

A technology of trypsin and trypsin gene, applied in the direction of microorganism-based methods, biochemical equipment and methods, peptidase, etc., can solve the problems of low trypsin activity and low protein expression

Active Publication Date: 2015-01-28
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The outstanding problems of heterologous expression of trypsin are low protein expression and low trypsin activity

Method used

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  • Method for optimizing signal peptide and improving exocytosis expression of trypsin
  • Method for optimizing signal peptide and improving exocytosis expression of trypsin
  • Method for optimizing signal peptide and improving exocytosis expression of trypsin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of recombinant plasmid

[0026] The trypsin gene of Streptomyces griseus (Gen Bank Accession No. M64471) is 672 bp in length. Design primers based on the nucleic acid sequences of the eight signal peptides. After two-step PCR, the signal peptide sequence and the gene sequence shown in SEQ ID NO.9 are homogenized to obtain the fusion gene fragment (using the trypsin gene shown in SEQ ID NO.9 as the template, the F2 and R primers are used for PCR and then the column is recovered. Then use the F1, R primer PCR column to recover the product to obtain the fusion fragment. The F1, F2, and R primer sequences are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12 respectively), according to figure 1 In the construction method shown, the fusion fragment and pPIC9K plasmid were digested with NotI and BamH I, and then ligated with T4 ligase overnight at 16°C after purification. The ligation product chemically transforms JM109 competent cells. The transf...

Embodiment 2

[0027] Example 2 Construction of trypsin-producing yeast engineering bacteria

[0028] The recombinant plasmid containing the recombinant gene with signal peptide added before the trypsin gene was linearized with Sal I, and electroporation was used to transform pichia pastoris GS115 competent cells. The specific method is as follows:

[0029] 1) Inoculate pichia pastoris GS115 activated on YPD plates in 25mL / 250mL Erlenmeyer flasks and culture overnight at 30℃; 1% inoculate the above-mentioned culture solution in 50mL / 500mL Erlenmeyer flasks, culture cell concentration OD600 is 1.3~1.5;

[0030] 2) Centrifuge at 5000r / min for 10min at 4℃ to collect the bacteria, and suspend the cells in 50mL and 25mL sterile water respectively;

[0031] 3) Resuspend the above cells in 5mL 1M sorbitol, centrifuge at 5000r / min for 10min at 4℃ to collect the bacteria;

[0032] 4) Resuspend the above cells in 500μL of 1M sorbitol, and aliquot 80μL / 1.5mL EP tubes for electrotransformation of competent cells;...

Embodiment 3

[0037] Example 3 Construction of recombinant bacteria

[0038] Using a two-step PCR method similar to Example 1, the nucleotide sequences such as the signal peptides of SEQ ID NO.1 to SEQ ID NO.8 (named as αas, Sas, Ipr, Iss, Lss, nsB, αmf, Kps) before fusion to the trypsin gene shown in SEQ ID NO.9, 8 recombinant genes αas-mt, Sas-mt, Ipr-mt, Iss-mt, Lss-mt, nsB-mt, αmf-mt, Kps-mt; respectively ligate the recombinant genes to the Pichia expression vector to obtain a recombinant plasmid; respectively electrotransform the recombinant plasmids into Pichia pastoris GS115 to obtain a recombinant yeast engineering strain.

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Abstract

The invention discloses a method for optimizing a signal peptide and improving exocytosis expression of trypsin, belonging to the technical field of genetic engineering. The signal peptide is fused with trypsin to express 8 types of pichia pastoris exocytosis signal peptides, and a starter (pAOX) is induced by methanol to express regrouped trypsin on pichia pastoris GS115 chromosome. Compared with the original alpha-factor signal peptide, the enzyme activity of the trypsin of the alpha mf signal peptide is improved by 2.75 times, and the problem that the exocytosis amount of the trypsin is low can be solved. By utilizing the method to produce the trypsin, the yield is high, the process is simplified, and the industrial application is convenient to realize.

Description

Technical field [0001] The invention relates to a method for optimizing signal peptides to increase the expression of extracellular secretion of trypsin, and belongs to the technical field of genetic engineering. Background technique [0002] Trypsin is an important type of serine protease that was discovered earlier and was first discovered in the digestive juice of mammals. Commercial trypsin is mostly extracted from mammalian pancreas. Because the pancreas is rich in proteases, it is difficult to separate and purify trypsin. In addition, most of the trypsin derived from mammals is a mixture of pancreatin, which has an immunogenic hazard to the human body in medical applications. [0003] In recent years, animal-derived trypsin has been expressed in E. coli and Pichia pastoris through genetic engineering. However, inclusion bodies often appear in E. coli expression. The expression of Pichia zymogen in pig and bovine trypsin requires exogenous enterokinase, Trypsin is activated ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/76C12R1/84
CPCC12N9/6427C12N15/81C12Y304/21004
Inventor 康振陈坚张云丰堵国成
Owner JIANGNAN UNIV
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