Pichia pastoris recombinant bacterium, culture method and application thereof

A Pichia pastoris and culture method technology, applied in the field of cultivation and Pichia pastoris recombinant bacteria, can solve the problems of high production cost, high technical difficulty, complicated separation and purification, etc., and achieve the effects of ensuring stability, low cost and simple process

Pending Publication Date: 2021-04-30
AURISCO PHARM(TIANJIN) INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is technically difficult, and the production cost is high, which is not conducive to large-scale production
[0004] CN110128552A, CN110498849A, CN107881187A, CN104592381A, CN108191981A, CN10724187A, CN107881187A disclose methods for expressing GLP-1 analogues in Escherichia coli. These methods will produce inclusion bodies, which require enzyme digestion and complicated separation and purification

Method used

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  • Pichia pastoris recombinant bacterium, culture method and application thereof
  • Pichia pastoris recombinant bacterium, culture method and application thereof
  • Pichia pastoris recombinant bacterium, culture method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0056] The process of constructing Pichia recombinant bacteria is as follows:

[0057] (1) Synthesis of Arg 34 GLP-1 （7-37） 、Arg 34 GLP-1 （9-37） Gene sequence: according to Pichia codon bias, for Arg 34 GLP-1 （7-37） 、Arg 34 GLP-1 （9-37） The original sequence was codon-optimized without changing the amino acid sequence to obtain the gene nucleotide sequence SEQ ID NO.2 and 3.

[0058] (2) Construction of recombinant plasmid: according to the above Arg 34 GLP-1 （7-37） 、Arg 34 GLP-1 （9-37） The full sequence of the gene and the multiple cloning site sequence on the Pichia pastoris expression vector pPinKα-HC (SEQ ID NO.1) is inserted into the plasmid insertion site map as shown in figure 1 Shown: gene fragment Arg 34 GLP-1 （7-37） 、Arg 34 GLP-1 （9-37） Ligated with the vector pPinKα-HC fragment to obtain the recombinant plasmid pPinKα-HC-Arg 34 GLP-1 （7-37） 、PinKα-HC-Arg 34 GLP-1 （9-37） ; The recombinant plasmid was sequenced, and the sequencing results showed tha...

Embodiment 2

[0064] The process of expression and isolation and purification of recombinant Pichia pastoris is as follows:

[0065] (1) Expression of recombinant Pichia pastoris: Inoculate the identified recombinant Pichia pastoris strains on a YPD plate and streak, and culture in an incubator at 30°C until a single colony grows. Take a single colony and inoculate it into fresh BMGY liquid medium, culture it in a shake flask at 30°C and 200rpm until OD600=2-4, collect the bacteria by centrifugation under sterile conditions, and resuspend the bacteria into fresh BMMY liquid with BMMY medium Add 1% methanol to the medium to induce OD600 of 1, and add once every 24 hours. After induction, samples of the supernatant of the medium were taken regularly, and the protein expression was detected by SDS-PAGE and liquid chromatography-mass spectrometry.

[0066](2) Separation and purification of recombinant Pichia pastoris after expression: transfer a single colony of recombinant bacteria containing...

Embodiment 3

[0070] The process of high-density fermentation of recombinant engineered bacteria is as follows:

[0071] (1) Strain activation: the monoclonal obtained from the PAD plate was inoculated into 50 m YPD sterile liquid medium, cultured at 30°C and 200 rpm for 48 h, and the first-grade seeds were obtained; the first-grade seeds were inoculated into five bottles of 100 ml YPD liquid culture medium (500mL shake flask), 30°C, 200 rpm for overnight culture to obtain secondary seeds; the secondary seeds were transferred to a 10L fermenter containing 5 L of fermentation medium at a 10% inoculation ratio, and fermented at 30°C in high density.

[0072] (2) High-density fermentation: a. Use ammonia water to adjust the pH of the medium to 4.0 before inoculation, and then add PTM1 at an amount of 4.35mL / L; in the fermentation medium. During the cultivation process, the dissolved oxygen in the fermenter first decreases and then increases. When the dissolved oxygen exceeds 80%, the carbon s...

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Abstract

The invention discloses a pichia pastoris recombinant bacterium, a culture method and an application thereof, the prepared and cultured recombinant yeast can efficiently express GLP-1 analogues, and then the GLP-1 analogues are efficiently prepared.

Description

technical field [0001] The invention belongs to the technical field of engineering bacteria, and in particular relates to a Pichia pastoris recombinant bacteria, a culture method and an application. Background technique [0002] Human glucagon-like peptide-1 (GLP-1) has medicinal potential in the treatment of type 2 diabetes. The multifaceted physiological actions of GLP-1 include stimulation of insulin expression, inhibition of glucagon secretion, and reduction of gastric emptying, contributing to the normalization of glucose levels. With the increasing prevalence of type 2 diabetes worldwide, many GLP-1 derivatives have been developed to enhance the biological activity and drug stability of GLP-1. Its growing demand calls for the development of bioprocesses utilizing recombinant microorganisms for the large-scale production of GLP-1 and its analogues. [0003] CN1225126A discloses a method for fermenting and expressing GLP-1 (7-37) by Saccharomyces cerevisiae through gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/16C12N15/81C07K14/605A61K38/26A61P3/10C12R1/84
CPCC07K14/605C12N15/815A61P3/10C12N2800/22A61K38/00
Inventor 彭志恩宋浩柳学伟巨晓芝杨晓瑜郭万成
Owner AURISCO PHARM(TIANJIN) INC
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