Pichia pastoris wall protein gcw28 and its surface display system and construction method

A construction method and surface display technology, applied in the field of Pichia pastoris wall protein Gcw28 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: 2011-12-14
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 gcw28 and its surface display system and construction method
  • Pichia pastoris wall protein gcw28 and its surface display system and construction method
  • Pichia pastoris wall protein gcw28 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 GCW28 gene

[0037] (1) Cloning of Pichia pastoris wall protein GCW28 gene

[0038] According to the gene sequence SEQ NO.4 of the Pichia pastoris wall protein GCW28 and the characteristics of multiple cloning sites on the Pichia pastoris plasmid pPIC9K, the 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 Not I restriction site. Using Pichia pastoris GS115 genomic DNA as template and P1 and P2 as primers, the wall protein GCW28 gene sequence was amplified by PCR. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; and then 30 cycles or less : Denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes; finally, extension at 72°C for 7 minutes....

Embodiment 2

[0045] Example 2: Construction of Pichia pastoris wall protein GCW28 surface display vector p9KGCW28

[0046] (1) Cloning of Pichia pastoris wall protein GCW28 gene

[0047] According to the Pichia pastoris wall protein GCW28 gene sequence and the characteristics of multiple cloning sites on the Pichia pastoris plasmid pPIC9K, the synthetic primers were designed:

[0048]

[0049] The underlined part of primer P2 is Not I restriction 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 template and P2 and P3 as primers, the wall protein GCW28 gene sequence was amplified by PCR. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; and then 30 cycles or less : Denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes; finally, extension at 72°C for 7 minutes. Obtain the Pichia pastoris wall protein GCW28 gene ...

Embodiment 3

[0052] Example 3: Construction of Pichia pastoris wall protein GCW28 surface display vector pZαAGCW28

[0053] (1) Cloning of Pichia pastoris wall protein GCW28 gene

[0054] According to the Pichia pastoris wall protein GCW28 gene sequence and the characteristics of multiple cloning sites on the Pichia pastoris plasmid pPIC9K, the synthetic primers were designed:

[0055] P2: 5’- TATATA GCGGCCGC TTAGATAGCCAAGAAGAG -3’ (SEQ NO: 3)

[0056] P4: 5’ -TATA CTCGAG GCCTTCCCTATATCTGA -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 template and P2 and P4 as primers, the wall protein GCW28 gene sequence was amplified by PCR. The amplification conditions were: pre-denaturation at 94°C for 5 minutes; and then 30 cycles: 94°C Denaturation for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes; finally, extension at 72°C fo...

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Abstract

The invention discloses Pichia pastoris wall protein, a surface display system constructed thereby and a construction method thereof. The amino acid sequence of the Pichia pastoris wall protein is SEQ NO:1. The Pichia pastoris wall protein cell surface display system is formed by using the Pichia pastoris wall protein Gcw28 as ankyrin and fixing target protein on the surface of the Pichia pastoris cell. The expression quantity of the wall protein Gcw28 in the Pichia pastoris is very high and is respectively 8 times and 18 times higher than that of the endogenesis ankyrin Pir1 protein and Pir2protein applied to the 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 Gcw28 and a constructed surface display system and construction method thereof. Background technique [0002] Microbial cell surface display technology is a technology that anchors proteins or peptides on the surface of cells. Microbial cell surface display system includes host bacteria, anchor protein and target protein, and sometimes a linker sequence is added between anchor protein and target protein. Microbial cell surface display has broad application prospects in polypeptide 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 microbial surface display systems mainly include phages, bacteria (such as E. coli Escherichia. coli Proteus mirabilis Proteus mirabilis Etc.), Saccharomyces cerevisiae ( ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/39C12N15/31C12N1/19C12N15/81C12R1/84
Inventor 林影张莉周新莹叶燕锐韩双艳郑穗平
Owner SOUTH CHINA UNIV OF TECH
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