A kind of production technology of recombinant trypsin

A production process, trypsin technology, applied in the field of recombinant trypsin purification production process, can solve the problems of low yield of recombinant trypsin, low renaturation rate of inclusion body, complicated purification process, etc.

Active Publication Date: 2021-06-01
杭州浦泰生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because this process is in the form of insoluble inclusion bodies, the renaturation rate of inclusion bodies is very low, and the purification process is relatively complicated, so the reported yield of recombinant trypsin is relatively low

Method used

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  • A kind of production technology of recombinant trypsin
  • A kind of production technology of recombinant trypsin
  • A kind of production technology of recombinant trypsin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] First, it is translated into a gene sequence (its sequence is shown in Seq ID No: 3) according to the porcine trypsinogen sequence (its sequence is shown in Seq ID No: 1), and the nucleic acid corresponding to the EK restriction site is contained at its 5' end sequence and 5'XhoI (ctcgag) restriction site, and a terminator and 3'NotI (gcggccgc) restriction site are added at the 3' end. The above sequence was synthesized by a gene synthesis service company, and cloned into the pGEM-T (promega company) vector. After sequencing verification, the sequence of the gene was complete and correct. Then extract the plasmids separately, use XhoI / NotI double enzymes to digest PGEMT to recover the porcine trypsin gene; use XhoI / NotⅠ double enzymes to digest pPICZαA yeast expression gene to recover a large fragment, and then connect the above two fragments to form a gene containing the yeast α-factor The recombinant yeast expression plasmid pPICZαA in the form of leader peptide-enter...

Embodiment 2

[0037] The expression fermentation of embodiment 2 porcine trypsin

[0038] Use the inoculation loop to pick the porcine trypsin engineering expression strain, put it into YPG medium (6 grams of tryptone, 3 grams of yeast powder, 6 grams of glycerol in a 5-liter glass beaker, set the volume to 300 milliliters with purified water, Packed in a 1000mL Erlenmeyer flask). Put the inoculated YPG shaker flask into a shaker, and cultivate it at 220rpm for about 18-24 hours, so that OD600≥5, as the seed solution.

[0039] Pour the seed liquid into the fermenter, supplement the yeast medium, and adjust the ventilation flow to not less than 10L / min. The condition control of the fermentation culture stage is as follows: the temperature is controlled at 20-30° C.; the pH is controlled at 4.0-5.0. Check and record every 0.5-2 hours. The records include: temperature, pH, dissolved oxygen PO2, stirring Stirr, ventilation Air, alkali volume, ventilation volume, and feed volume. When cultiva...

Embodiment 3

[0041] The purification of embodiment 3 porcine trypsin

[0042] 1) Sample pretreatment: add 1 times of purification water to the fermentation supernatant, and the conductance of the final sample solution is about 7.0ms / cm; the pH is controlled at about 4.5.

[0043] 2) Chromatographic column equilibrium: a chromatographic column with a diameter of 25 cm, and the filler is an ion exchange column SP Bestarose FF (Borgeron (Shanghai) Biotechnology Co., Ltd.). Use equilibration buffer (50mM NaAc-HAc, 2mM CaCl2, pH4.5;) to equilibrate 3CV, conductance and pH baseline go flat.

[0044] 3) Sample loading: take the pretreated fermentation broth and load the sample.

[0045] 4) Chromatographic column re-equilibration: equilibrate 2CV with equilibration buffer.

[0046] 5) Sample elution: elute the target peak with eluent (50mM NaAc-HAc, 0.4M NaCl, 2mM CaCl2, pH4.5).

[0047] 6) Chromatographic column regeneration: first wash 1CV with 1mol / L NaOH, then wash 1CV with eluent, and then...

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Abstract

The invention provides a production process for expressing soluble recombinant trypsin by using yeast, so that trypsin can be highly expressed in the supernatant, and the recombinant trypsinogen can be purified from the supernatant of the fermentation broth by ion exchange chromatography , the highly active recombinant trypsin obtained by autocatalysis is far superior to the Escherichia coli expression process in terms of cost, yield, activity, purity and other technical indicators, so it is of great industrial production value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a purification and production process of recombinant trypsin. Background technique [0002] Pancreatin is a digestive drug recorded in the pharmacopoeias of various countries. It is a mixture of various enzymes extracted from animal pancreas. The main components are trypsin, pancreatic lipase and pancreatic amylase. According to the provisions of the Chinese Pharmacopoeia 2015 edition, this strain is a mixture of various enzymes extracted from pig, sheep or bovine pancreas, mainly trypsin, pancreatic amylase and pancreatic lipase. Calculated as a dry product, trypsin activity per lg should not be less than 600 units, pancreatic amylase activity should not be less than 7000 units, and pancreatic lipase activity should not be less than 4000 units. At present, the production of pancreatin is mainly extracted from animal pancreas, and the existing pancreatin production process is used t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64C12N15/81
CPCC12N9/6408C12N15/815C12Y304/21004
Inventor 乐峰松
Owner 杭州浦泰生物科技有限公司
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