Pichia yeast expression system without carbon source repression, and establishing method and applications

A carbon source-free, yeast-based technology, applied in the field of bioengineering, can solve the problems of PAOX1 single regulation mode, carbon source repression, etc.

Active Publication Date: 2018-12-07
EAST CHINA UNIV OF SCI & TECH
View PDF4 Cites 11 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although non-methanol-induced P AOX1 The research has made great breakthroughs, but still can not

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Pichia yeast expression system without carbon source repression, and establishing method and applications
  • Pichia yeast expression system without carbon source repression, and establishing method and applications
  • Pichia yeast expression system without carbon source repression, and establishing method and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1. Methanotrophic yeast constitutive expression system without carbon source repression

[0077] 1. Construction of pPlacO1cAG plasmid

[0078] Using lacO-cAOX1F (SEQ ID NO: 14) and pPcAG R (SEQ ID NO: 15) as primers and pPcAG F (SEQ ID NO: 16) and lacO-pPIC R (SEQ ID NO: 17) as primers, respectively, The AOX1 core promoter (ie, SEQ ID NO: 7) and GFP regions, as well as the pPHis integration site and resistance fragment region were amplified from the pPAG plasmid by PCR, and the two fragments were assembled by a seamless cloning kit , the resulting recombinant plasmid was pPlacO1cAG.

[0079] 2. Screening of electroporated Pichia pastoris and GS115-lacO1cAG strain

[0080] The recombinant plasmid pPlacO1cAG was electrotransformed into Pichia pastoris strain GS115, spread on a YND plate without histidine, and cultured in a 30°C incubator for 48-72 hours. Pick the single clone grown on the plate into the liquid medium, and extract the genome after culturing on ...

Embodiment 2

[0093] Example 2, Inducible Regulatory Expression System

[0094] 1. Construction of an inducible promoter expressing LacIMit1AD fusion polypeptide plasmid

[0095] Using LacIF (SEQ ID NO: 26) and pGout R (SEQ ID NO: 27) as primers, the ORF frame, terminator and resistance region fragments of the LacIMit1AD fusion polypeptide were amplified from pGGLacIMit1AD by PCR method, respectively, and The MSC1 promoter, MAL31 promoter, and GAL4 promoter fragments amplified from the Pichia pastoris GS115 genome were seamlessly assembled using a seamless cloning kit, so that the above promoters were located upstream of the fusion polypeptide ORF box and could drive the polypeptide Express. Recombinant plasmid pGP MSC LacIMit1AD, pGP MAL LacIMit1AD, pGP GAL LacIMit1AD.

[0096] MSC1 promoter amplification primers: pG-MSC F (SEQ ID NO:28) and LacI-MSC R (SEQ ID NO:29);

[0097] MAL31 promoter amplification primers: pG-MAL F (SEQ ID NO:30) and LacI-MAL R (SEQ ID NO:31);

[0098] GAL4 ...

Embodiment 3

[0104] Example 3, Methanotrophic yeast expression system containing different lacO copy numbers

[0105] 1. Screening of Pichia pastoris GS115-LM single-copy strain

[0106] The recombinant plasmid pGGLacIMit1AD was electrotransformed into GS115 strain, spread on the YPD solid medium plate supplemented with Zeocin antibiotic, and cultured in a 30°C incubator for 48-72 hours. Pick the single clone grown on the plate into the liquid medium, and extract the genome after culturing on a shaker at 30°C, and verify the LacI copy number by real-time PCR. The expression strains of Pichia pastoris that were detected as single copy by real-time PCR were named GS115-LM respectively.

[0107] 2. Construction of plasmids containing different copy numbers of lacO

[0108] The recombinant plasmid pPlacO1cAG (containing 1 copy of lacO) was linearized by double enzyme digestion in SacI / XhoI by enzyme digestion, and then ligated with the lacO fragment to obtain the recombinant plasmid pPlacO2c...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to an enhanced methylotrophic pichia yeast expression system without carbon source repression, and an establishing method and applications, wherein the pichia yeast expression system is high in expression amount. The invention discloses a method capable of eliminating methanol inducible promoter single methanol carbon source dependence and carbon source severe repression; artificial modification of the transcription regulation and control heredity route in methylotrophic yeast cells is adopted, promoter transcription intensity is increased, and the regulation and controlmode is changed. The yeast expression system and the expression method disclosed in the invention are capable of eliminating dependence of promoters which are dependent on methanol induction and are influenced by carbon source repression on methanol, so that high efficiency expression of exogenous polypeptides can be realized at other carbon source conditions, the highest expression amount is 5 times of that of wild type AOX1 promoter at methanol conditions.

Description

technical field [0001] The invention belongs to the field of bioengineering; more specifically, the invention relates to a carbon-source-free suppression Pichia expression system, its establishment method and application. Background technique [0002] In recent years, the proportion of biological products in people's production and life has been increasing, and most biological products such as vaccines, antibodies, antimicrobial peptides, antibiotics, etc. are almost all produced through heterologous expression. In the process of using model organisms to express biological products heterologously, the transcription initiation process guided by the promoter is a key step in protein expression. Therefore, a strong and regulatable promoter has always been an essential tool for high-level expression of foreign proteins. [0003] Alcohol oxidase (Aox1) promoter in Pichia pastoris (P AOX1 ) is currently the most powerful promoter, which has been used to express a variety of fore...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12P21/02C12N1/19C12N15/62C12R1/84C12R1/78C12R1/72C12R1/645
CPCC12P21/02C07K14/39C07K2319/80
Inventor 蔡孟浩刘启周祥山张元兴
Owner EAST CHINA UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap