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

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...

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, and difficult to select an optimized enzyme with the desired properties, which cannot meet the actual needs of protein product production.

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