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Method for high-efficiency expression of carboxypeptidase Y derived from actinomucor elegans

A technology of Mucor radiatae and carboxypeptidase, applied in the field of bioengineering, can solve the problems of unsuitability for industrial applications, low protein expression level of carboxypeptidase Y, and the protein expression level needs to be improved, etc.

Active Publication Date: 2018-03-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The carboxypeptidase Y that is currently studied is mainly derived from Saccharomyces cerevisiae, Pichia pastoris and fission yeast, among which the carboxypeptidase Y derived from Saccharomyces cerevisiae is the most fully studied, while the carboxypeptidase Y derived from Mucor is very rare. rarely reported
Moreover, the protein expression level of carboxypeptidase Y derived from Mucor is not high and not suitable for industrial application. Therefore, the protein expression level of carboxypeptidase Y derived from Mucor needs to be improved

Method used

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  • Method for high-efficiency expression of carboxypeptidase Y derived from actinomucor elegans
  • Method for high-efficiency expression of carboxypeptidase Y derived from actinomucor elegans
  • Method for high-efficiency expression of carboxypeptidase Y derived from actinomucor elegans

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 Construction of recombinant plasmid pPICZα-procpy

[0033] The primers were designed with the laboratory-preserved plasmid pPIC9K-procpy, and the EcoRI restriction site and protective base were introduced upstream, the Not I restriction site and protective base were introduced downstream, and the His tag was introduced at the C-terminal.

[0034] Using pPIC9K-procpy as a template, the full-length procpy gene containing EcoR I, Not I restriction sites and C-terminal His tag was obtained by PCR. The amplified procpy gene product was digested with EcoR I and Not I and connected to pPICZα digested with the same restriction endonuclease to obtain the recombinant plasmid pPICZα-procpy, transformed into Escherichia coli JM109, and sequenced to check whether the sequence was correct .

Embodiment 2

[0035] Example 2 Construction of recombinant Pichia pastoris GS115 / pPICZα-procpy

[0036] The recombinant plasmid pPICZα-procpy with the correct sequence was successfully constructed, digested with the restriction endonuclease Sac I, set the electroporation parameters: 1500v, 400Ω, 4-6ms, electrotransformed Pichia pastoris GS115, and coated with zeocin 100μg / mL Place the YPD plate in an incubator at 28°C for 2-3 days to screen for recombinant strains.

Embodiment 3

[0037] Example 3 Screening of High Copy Recombinant Strains

[0038] Put the transformant on the YPD plate containing zeocin 100 μg / mL, use a sterilized pipette tip to spot on the YPD plate containing zeocin 600, 1000, 1800 μg / mL in sequence, and place it in an incubator at 28°C for 2 -3 days, transformants were screened. The recombinant strain that can grow on the YPD plate containing zeocin 100 μg / mL but cannot grow on the YPD plate containing zeocin 600 μg / mL is named zeocin-100. In this way, zeocin-600, zeocin-1000, zeocin- 1800.

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Abstract

The invention discloses a method for high-efficiency expression of carboxypeptidase Y derived from actinomucor elegans and belongs to the field of bioengineering. By construction of expression recombinant plasmid pPICZalpha-procpy and electrotransformation of pichia yeast GS115 and through a method for continuously raising antibiotic, expression recombinant strains with different copy numbers arescreened out; then, chaperone BIP derived from pichia yeast is inoculated into the recombinant strain so as to finally obtain an expression recombinant strain for high-efficiency expression of carboxypeptidase Y derived from actinomucor elegans; and after 6 days of induced expression, expression level reaches 525 + / - 20 mg / L.

Description

technical field [0001] The invention relates to a method for efficiently expressing carboxypeptidase Y derived from Radimucor elegans, belonging to the technical field of bioengineering. Background technique [0002] Carboxypeptidase Y (carboxypeptidase Y) is an exopeptidase that can hydrolyze the C-terminal amino acid residues of peptide chains. It has a wide substrate spectrum and has certain activity on proline residues that are difficult for other carboxypeptidases to degrade. , so it can be used as a tool enzyme in the detection process of polypeptide chain sequencing and mass spectrometry detection, and it also has certain applications in the removal of bitterness of protein hydrolyzate. [0003] The carboxypeptidase Y that is currently studied is mainly derived from Saccharomyces cerevisiae, Pichia pastoris and fission yeast, among which the carboxypeptidase Y derived from Saccharomyces cerevisiae is the most fully studied, while the carboxypeptidase Y derived from Mu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N9/48C12R1/84
CPCC12N9/485C12Y304/16005
Inventor 周哲敏刘中美周丽崔文璟张欢欢郭军玲陈昶旭
Owner JIANGNAN UNIV
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