Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Pichia pastoris wall protein gcw12 and its surface display system and construction method

A construction method and surface display technology, applied in the field of Pichia pastoris wall protein Gcw12 and its constructed surface display system, can solve the problems of low expression of Pir protein and general effect of anchoring protein, etc., and achieve high expression efficiency Effect

Active Publication Date: 2011-11-30
SOUTH CHINA UNIV OF TECH
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inventors found that the expression level of the Pir protein itself is low, and the effect of the anchor protein as the surface display system of Pichia pastoris is general

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 pastoris wall protein gcw12 and its surface display system and construction method
  • Pichia pastoris wall protein gcw12 and its surface display system and construction method
  • Pichia pastoris wall protein gcw12 and its surface display system and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Cloning, expression and identification of Pichia pastoris wall protein GCW12 gene

[0037] (1) Cloning of Pichia pastoris wall protein GCW12 gene

[0038] According to the gene sequence SEQ NO.4 of the Pichia pastoris wall protein GCW12 and the characteristics of the multiple cloning site on the Pichia pastoris plasmid pPIC9K, synthetic primers were designed:

[0039]

[0040] The box part of primer P1 is EcoR I restriction site, the underlined part is Mlu I restriction site, the italic part is the FLAG tag sequence; the underlined part of primer P2 is notI restriction site. Using Pichia pastoris GS115 genomic DNA as a template and P1 and P2 as primers, the wall protein GCW12 gene sequence was amplified by PCR. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; followed by 30 more cycles : Denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute; finally extension at 72°C for 7 m...

Embodiment 2

[0045] Example 2: Construction of Pichia pastoris wall protein GCW12 surface display vector p9KGCW12

[0046] (1) Cloning of Pichia pastoris wall protein GCW12 gene

[0047] According to the gene sequence of Pichia pastoris wall protein GCW12 and the characteristics of multiple cloning sites on Pichia pastoris plasmid pPIC9K, synthetic primers were designed:

[0048]

[0049] The underlined part of primer P2 is not I enzyme cleavage site; the box part of primer P3 is EcoR I restriction site, the underlined part is Mlu I restriction site. Using Pichia pastoris GS115 genomic DNA as a template and P2 and P3 as primers, the wall protein GCW12 gene sequence was amplified by PCR. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; followed by 30 cycles or less : Denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute; finally extension at 72°C for 7 minutes. The Pichia pastoris wall protein GCW12 ...

Embodiment 3

[0052] Example 3: Construction of Pichia pastoris wall protein GCW12 surface display vector pZαAGCW12

[0053] (1) Cloning of Pichia pastoris wall protein GCW12 gene

[0054] According to the gene sequence of Pichia pastoris wall protein GCW12 and the characteristics of multiple cloning sites on Pichia pastoris plasmid pPIC9K, synthetic primers were designed:

[0055] P2: 5'-TATTAT GCGGCCGC CTAGATCAGACCAACAGC-3' (SEQ NO: 3)

[0056] P4: 5’ – ATAT CTCGAG ACTCCACCGGCTTGCCT-3' (SEQ NO: 6)

[0057] The underlined part of primer P2 is not I restriction site; the underlined part of primer P4 is xho I restriction site. Using Pichia pastoris GS115 genomic DNA as a template, and P2 and P4 as primers, the wall protein GCW12 gene sequence was amplified by PCR method. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; followed by 30 cycles: 94°C Denaturation for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute; final extens...

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 discloses a Pichia pastoris wall protein, a surface display system constructed by the same, and a construction method. The amino acid sequence of the Pichia pastoris wall protein is shown as SEQNO:1; and the Pichia pastoris cell surface display system is prepared by fixing a target protein on the Pichia pastoris cell surface through the Pichia pastoris wall protein GCW12 serving as an anchor protein. The expression level of the wall protein GCW12 in Pichia pastoris is high, and is 11 times and 24 times higher than those of endogenous anchor proteins Pir1 and Pir2 in the conventional Pichia pastoris surface display system.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Pichia pastoris wall protein Gcwl2 and its constructed surface display system and construction method. Background technique [0002] Microbial cell surface display technology is a technology that fixes proteins or polypeptides on the cell surface through anchor proteins. The microbial cell surface display system includes host bacteria, anchor proteins and target proteins, and sometimes a linker sequence is added between the anchor proteins and target proteins. Microbial cell surface display has broad application prospects in peptide separation, whole-cell catalysts, whole-cell adsorbents, vaccine and antibody production, protein library screening, biosensors, and bioremediation. [0003] At present, the host bacteria that are widely used in the microbial surface display system mainly include phages, bacteria (such as Escherichia coli Escherichia. coli Proteus mirabilis Proteus ...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/39C12N15/31C12N15/81C12N1/19
Inventor 林影周新莹张莉叶燕锐韩双艳郑穗平
Owner SOUTH CHINA UNIV OF TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com