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Pichia pastoris wall protein gcw3 and its surface display system and construction method

A construction method and surface display technology, which are applied in the field of Pichia pastoris wall protein Gcw3 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

Inactive Publication Date: 2011-11-30
SOUTH CHINA UNIV OF TECH
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  • 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

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  • Pichia pastoris wall protein gcw3 and its surface display system and construction method
  • Pichia pastoris wall protein gcw3 and its surface display system and construction method
  • Pichia pastoris wall protein gcw3 and its surface display system and construction method

Examples

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Effect test

Embodiment 1

[0036] Example 1: Cloning, expression and identification of Pichia pastoris wall protein GCW3 gene

[0037] (1) Cloning of Pichia pastoris wall protein GCW3 gene

[0038] According to the gene sequence SEQ NO.4 of Pichia pastoris wall protein GCW3 and the characteristics of multiple cloning sites 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 GCW3 gene sequence was amplified by PCR method. 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 mi...

Embodiment 2

[0045] Example 2: Construction of Pichia pastoris wall protein GCW3 surface display vector p9KGCW3

[0046] (1) Cloning of Pichia pastoris wall protein GCW3 gene

[0047] According to the gene sequence of Pichia pastoris wall protein GCW3 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 the genomic DNA of Pichia pastoris GS115 as a template, and using P2 and P3 as primers, the wall protein GCW3 gene sequence was amplified by PCR method. 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 wal...

Embodiment 3

[0052] Example 3: Construction of Pichia pastoris wall protein GCW3 surface display vector pZαAGCW3

[0053] (1) Cloning of Pichia pastoris wall protein GCW3 gene

[0054] According to the gene sequence of Pichia pastoris wall protein GCW3 and the characteristics of multiple cloning sites on Pichia pastoris plasmid pPIC9K, synthetic primers were designed:

[0055]

[0056] 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 GCW3 gene sequence was amplified by PCR method. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; followed by 30 or less cycles: 94°C Denaturation for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute; final extension at 72°C for 7 minutes. The Pichia pastoris wall protein GCW3 gene fragment was obtained.

[0057] (2) Construction o...

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Abstract

The invention discloses a Pichia pastoris wall protein, a surface display system constructed by the Pichia pastoris wall protein, 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 constructed by fixing a target protein on the surface of the Pichia pastoris cell by using the Pichia pastoris wall protein Gcw3 as an anchor protein. The expression level of the Pichia pastoris wall protein Gcw3 is high, and is respectively 6 times and 14 times higher than those of ndogenous 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 Gcw3 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

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Application Information

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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
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