Method for rapidly constructing protein mutant pichia pastoris expression library and application thereof

A Pichia pastoris and protein technology, which is applied in the field of protein engineering, can solve the problems of not meeting the actual needs of protein products, low work efficiency, and difficulty in directional selection and optimization of enzymes.

Active Publication Date: 2021-06-08
HENAN ACAD OF SCI INST OF BIOLOGY LIABILITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing error-prone PCR random mutation method to construct a mutant library is complex in operation, low in work efficiency,

Method used

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  • Method for rapidly constructing protein mutant pichia pastoris expression library and application thereof
  • Method for rapidly constructing protein mutant pichia pastoris expression library and application thereof
  • Method for rapidly constructing protein mutant pichia pastoris expression library and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] A method for rapidly constructing a protein mutant Pichia pastoris expression library of the present invention comprises the following steps:

[0021] (1) Construct the target gene into the Pichia pastoris vector pGAPZα series vector, and construct the plasmid:

[0022] Taking an artificially synthesized α-galactosidase gene GalA of vibrio fischeri (fecheri) as the target gene, the sequence is SEQ1, using DNA restriction endonuclease EcoRI / NotⅠ to pair gene GalA and Pichia pastoris constitutive expression vector pGAPZαA was double-digested at the same time, and then the gene GalA was ligated into the carrier plasmid pGAPZαA with T4 DNA ligase to form the expression plasmid pGAPZαA-GalA, and the plasmid was digested with DNA restriction endonuclease EcoRI / NotⅠ to ensure the construction of the plasmid Success, after digestion, there are plasmid pGAPZαA bands and GalA DNA bands, and the plasmid construction is successful;

[0023] SEQ1:

[0024] TTGGTTAGACCAGGTAACGTTGGT...

Embodiment 2

[0035] A method for rapidly constructing a protein mutant Pichia pastoris expression library of the present invention comprises the following steps:

[0036] (1) Construct the target gene into the Pichia pastoris vector pGAPZα series vector, and construct the plasmid:

[0037] Taking an artificially synthesized α-galactosidase gene GalB of vibrio fischeri (fecheri) as the target gene, the sequence is SEQ2, using DNA restriction endonuclease EcoRI / NotⅠ to pair gene GalB and Pichia pastoris constitutive expression vector pGAPZαA was double-digested at the same time, and then the gene GalB was ligated into the vector pGAPZαA with T4 DNA ligase to form the expression plasmid pGAPZαA-GalB, and the plasmid was digested with DNA restriction endonuclease EcoRI / NotⅠ to ensure the successful construction of the plasmid , after digestion, there are plasmid pGAPZαA bands and GalB DNA bands, and the plasmid construction is successful;

[0038] SEQ2:

[0039] TTGAACAACGGTTTGGCTAGAACTCCACA...

Embodiment 3

[0050] A method for rapidly constructing a protein mutant Pichia pastoris expression library of the present invention comprises the following steps:

[0051] (1), the target gene is constructed into the Pichia pastoris vector pGAPZα series vector, and the plasmid is constructed;

[0052]Using a synthetic glucose oxidase gene GOD of Aspergillus niger as the target gene, the sequence is SEQ3, and the gene GalB and the Pichia pastoris constitutive expression vector pGAPZαA are simultaneously doubled with DNA restriction endonuclease KpnⅠ / NotⅠ. Digest, and then use T4 DNA ligase to connect the gene GOD into the vector pGAPZαA to form the expression plasmid pGAPZαA-GOD, and use the DNA restriction endonuclease KpnⅠ / NotⅠ to check the plasmid to ensure the successful construction of the plasmid. After cutting, there are plasmid pGAPZαA bands and GOD DNA bands, and the plasmid construction is successful;

[0053] SEQ3:

[0054] AGCAATGGCATTGAAGCCAGCCTCCTGACTGATCCCAAGGATGTCTCCGGCCGCA...

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Abstract

The invention relates to a method for rapidly constructing a protein mutant pichia pastoris expression library and application thereof, which can effectively solve the problems of rapidly constructing the protein mutant pichia pastoris expression library and realizing directional selection of required property optimization enzyme, and the method comprises the following steps: constructing a target gene into a pichia pastoris vector pGAPZalpha series vector to construct plasmids, carrying out a polymerase chain reaction to obtain a PCR product, adding saturated sodium acetate and ethanol into the PCR product, carrying out centrifugation, carrying out precipitation and drying at room temperature, adding sterile deionized water for dissolving, electrically transforming the PCR product into pichia pastoris GS115 competent cells according to a yeast transformation method, coating a YPDS culture medium plate containing bleomycin with the cells to screen positive clones, extracting yeast genome DNA, then sequencing the PCR product, calculating the base mutation rate and gene mutation rate, and realizing the rapid construction of protein mutant Pichia pastoris expression library. The method has the advantages that the method is easy to operate, the time and labor are saved, the working efficiency is high, the application range is wide, and the economic and social benefits are huge.

Description

technical field [0001] The invention relates to the field of protein engineering, in particular to a method for rapidly constructing a protein mutant Pichia pastoris expression library and its application. Background technique [0002] Protein products are widely used in life and production, and the application of various enzyme preparations is the most prominent, including detergent reagents, feed enzyme preparations, food additives, scientific research tool enzymes, papermaking enzymes, environmental protection enzymes, and medical enzymes Etc., in order to obtain enzymes with superior properties and more in line with the needs of practical applications, it is necessary to artificially mutate the enzymes from natural sources to change the thermal stability, pH tolerance, protease tolerance, unit activity value and other characteristics of the enzyme. Mutation methods include site-directed design mutation and in vitro directed evolution. Error-prone PCR is a commonly used r...

Claims

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Application Information

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IPC IPC(8): C12N15/10C40B50/06C12N15/81C12N9/24C12N9/40C12N9/04
CPCC12N15/1086C40B50/06C12N15/815C12N9/2465C12Y302/01022C12Y101/03004C12Y302/01025C12N9/2402C12N9/0006C12Q2531/113
Inventor 刁文涛权淑静李珊珊陈晓飞刘德海杨文玲雷高周伏忠张宗源陈国参王继雯胡虹李寒冰
Owner HENAN ACAD OF SCI INST OF BIOLOGY LIABILITY
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