Non-methanol evoked pichia pastoris protein expression system and use thereof

Abstract

The invention belongs to biological technical field and relates to the improvement technology and the application of the expression system of existing pichia pastoris. Based on the metabolic pathway and the inherited characteristics of pichia pastoris carbon source and nitrogen source, and the clone and analysis to the formaldehyde dehydrogenase 1(FLD1) PFLD1 promoter gene which can be expressed under the induction of non-toxic nitrogen source nutrient such as choline, the invention provides a pichia pastoris expression vector whose promoter is a formaldehyde dehydrogenase promoter, the preparation method and the application as well as the corresponding expression system and the application of the expression system. The expression vector or expression system of the invention can take non-toxic choline nitrogen source as inducer when expressing foreign protein, with nearly the same foreign protein expression efficiency as taking toxic methanol as inducer. The green and safe expression system which does not use toxic inducer can be applied to produce genetic engineering food and pharmaceutical genetic engineering products.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a Pichia pastoris expression system capable of inducing the production of exogenous proteins by using non-toxic nitrogen source components and its application in the production of pharmaceutical and food-grade proteins. Background technique [0002] The gene expression system is an important content in the field of molecular biology and life science research, and it is also an important bioreactor necessary for the development of genetic engineering drugs and vaccines. Escherichia coli (E.coli) has been used as a host bacteria for expressing foreign genes, and has successfully expressed a variety of foreign proteins, but it cannot express proteins with complex structures, and the yield of secreted expression is low, while inclusion There are still many problems in the complete renaturation of the expressed product in vivo, and the expressed product is often accompanied by ...

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Problems solved by technology

[0003] Saccharomyces cerevisiae (S.cerevisiae) was first used as an expression host for exogenous genes. However, people found that S.cerevisiae expressed recombinant proteins had its limitations: (1) the yield was usually low; (2) The expression plasmid is easy to be lost, and the integration of foreign genes is unstable; (3) lack of strong and strictly regulated promoters; (4) the excessive glycosylation of foreign proteins significantly enhances the antigenicity, which is not suitable for therapeutic use; (5) poor secretion efficiency, especially exogenous proteins with a molecular weight greater than 30kDa are hardly secreted, complicating downstream purification; (6) not suitable for high-density fermentation and difficult for industrial production

So far, although most proteins have been AOX1 The promoter was successfully expressed, but in practical applications, the promoter brought many problems or inconveniences in many cases. For example, in shake flask culture, methanol is very easy to evaporate quickly, which is not conducive to the control of methanol concentration, and It is necessary to add methanol to the medium several times, and it is not enough to induce expression when the amount is small. It is reported that high concentration of methanol will inactivate 99% of AOX1 (alcohol oxidase 1, alcohol oxidase 1) of Mut+ yeast strain and cause cell poisoning and die

At the same time, it may also cause a large amount of toxic methanol gas to diffuse in the laboratory and surrounding environment; for example, when using recombinant Pichia pastoris (P. Inflammable and explosive methanol is undoubtedly a big safety hazard; in addition, the exogenous protein expressed by recombinant Pichia pastoris (P. pastoris) is often accompanied by a certain amount of methanol, because methanol is harmful to humans and animals. It has high toxicity, so in the purification process of the target protein, the problem of methanol residue and demethanolization has to be considered, especially for drugs and foods produced by methanol-induced expression. According to the US FDA (U.S. Food and Drugs Administration) standard, usually It is considered to be unsafe, namely NO GRAS (generally regarded as safe)

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