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Method for improving trypsin activity through artificially-designed self-activated leading peptide sequence

A trypsin and leader peptide technology, applied in the field of genetic engineering, can solve the problems of low expression, toxicity, and low activation efficiency in vitro

Active Publication Date: 2017-06-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of trypsin in the form of zymogen has the problems of low activation efficiency in vitro and high production cost
The expression of mature trypsin sequence also has the following problems: trypsin, as a protease, has protease toxicity to the expression host during the secretion and expression process; and then triggers intracellular degradation, resulting in low expression levels

Method used

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  • Method for improving trypsin activity through artificially-designed self-activated leading peptide sequence
  • Method for improving trypsin activity through artificially-designed self-activated leading peptide sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of recombinant plasmid pPIC9K-Sedeif containing recombinant trypsin gene (Sedeif)

[0024] Using the nucleic acid sequence shown in sequence 3 in the patent CN 104328102 A as a template, R primer (sequence shown in SEQ ID NO.8) and F primer1 (sequence shown in SEQ ID NO.9) as primers, perform PCR to obtain a PCR product . Using the PCR product as a template, Rprimer (sequence such as SEG ID NO.8) and F primer2 (sequence such as SEQ ID NO.10) as primers, the nucleotide sequence shown in SEQ ID NO.7 was obtained and named as Sedeif.

[0025] Fusion PCR technology was used to fuse the signal peptide sequence shown in SEQ ID NO.5 at the 5' end of the Sedeif sequence. The fusion fragment and pPIC9K were digested with Not I and BamH I respectively, and purified with T4 ligase overnight at 16°C connect. The ligation product was chemically transformed into JM109 competent cells. The transformation solution was coated with kanamycin (50mg / L) LB plates, ...

Embodiment 2

[0026] Example 2 Construction of high-yielding mature trypsin yeast engineering bacteria

[0027] The Pichia pastoris expression plasmid pPIC9K-Sedeif was linearized with SalI, and the Pichia pastorisGS115 competent cells were transformed by electroporation. The specific method is as follows:

[0028] 1) Inoculate Pichia pastoris GS115 activated on YPD plate in 25mL / 250mL Erlenmeyer flask, and culture overnight at 30°C; inoculate 1% of the above culture solution into 50mL / 500mL Erlenmeyer flask, and the cultured bacteria concentration OD600 is 1.3-1.5;

[0029] 2) Centrifuge at 5000r / min at 4°C for 10min to collect the cells, suspend the cells in 50mL and 25mL of sterile water respectively; 3) resuspend the above cells in 5mL of 1M sorbitol, centrifuge at 5000r / min at 4°C for 10min to collect the cells;

[0030] 4) Resuspend the above cells in 500 μL of 1M sorbitol, aliquot into 80 μL / 1.5mL EP tubes for electrotransformation of competent cells;

[0031] 5) Mix 20 μL of linear...

Embodiment 3

[0035] Example 3 Recombinant Pichia pastoris 3L tank fermentation

[0036] The engineering bacterium constructed in Example 2 is used as the production strain (the recombinant bacterium constructed in the patent CN 104328102 A is used as the control strain), after the activation of the YPD plate, inoculate 50mL / 250mL seed medium, and cultivate it at 30°C for 24h at 220r / min as Fermentation culture seed liquid. 10% was inoculated with 800mL / 3L fermentation medium, pH 5.5, cultured in stages at 30°C: 0-19h, 500rmp / min culture, dissolved oxygen dropped from 100% to about 8%, and then increased to about 60%; 19-34h, The rotational speed gradually increased to 1000rmp / min, and 50% glycerol was fed exponentially, DO began to drop to about 7%, and then rose to 79.1%; 34-168h, 1.8% (V / V) methanol was fed to induce trypsin production.

[0037] Seed medium (g L –1 ): Peptone 20, Yeast Extract 10, Glucose 20.

[0038] Fermentation medium (g L –1 ): Glycerol 40; K 2 SO 4 18; KOH 4.1...

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Abstract

The invention discloses a method for improving trypsin activity through an artificially-designed self-activated leading peptide sequence and belongs to the field of genetic engineering. Trypsin of an artificial self-activated leading peptide sequence TPAPPSDDLGTFDDDDK is fused at an expression N end of a pichia pastoris engineered strain constructed by the method. Trypsin activity (trypsin amidase activity) of the yeast engineered strain GS115-Sedeif reaches 156U.mL-1, and esterase activity is 15015.8U.mL-1 (BAEE serving as a substrate), which are 1.82 times and 3.29 times of strain trypsin activity before modification. By the method, the key problem of low trypsin heterogeneous expression is solved. By applying the recombinant strain to produce trypsin, the method has the advantages of high yield, simplified fermentation process and convenience in industrial application.

Description

technical field [0001] The invention relates to a method for artificially designing a self-activating leader peptide sequence to improve trypsin enzyme activity, which belongs to the technical field of genetic engineering. Background technique [0002] As a specific polypeptide hydrolase, trypsin has been widely used in many fields. As an important enzyme preparation for leather processing, it is used in local treatment of leather and deliming and softening: removal of scale in bare hide, removal of fiber matrix, and loose collagen, thereby enhancing the plumpness, elasticity, and smoothness of the leather; application In medicine, it can accelerate the purification of wounds, promote the regeneration of granulation tissue, anti-inflammation, treat snake venom bites, digestive tract diseases, etc.; specifically hydrolyze natural proteins such as collagen to prepare functional polypeptides; trypsin is also used in frozen food processing, polypeptides mass spectrometry etc. ...

Claims

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Application Information

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IPC IPC(8): C12N9/76C12N1/19C12N15/81C12R1/84
CPCC07K2319/02C12N9/6427C12N15/625C12N15/815C12Y304/21004
Inventor 康振张云丰陈坚堵国成刘松
Owner JIANGNAN UNIV
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