CN105039471B

CN105039471B - Method for improving expression quantity of methanol nutritional yeast expression system

Info

Publication number: CN105039471B
Application number: CN201510175618.6A
Authority: CN
Inventors: 宁荣良; 焦策; 张宏达; 封海生; 王超; 黄成潭; 李汉仁
Original assignee: Yichang Hec Changjiang Pharmaceutical Co ltd
Current assignee: Yichang Hec Changjiang Pharmaceutical Co ltd
Priority date: 2014-04-18
Filing date: 2015-04-14
Publication date: 2022-11-18
Anticipated expiration: 2035-04-14
Also published as: CN105039471A

Abstract

The invention provides a method for improving expression quantity of a methanol nutritional yeast expression system. Protease inhibitors such as benzamidine hydrochloride are added into methanol and fed-batch fermentation system is carried out in the fermentation induction stage, so that degradation of recombinant protein by thallus autoprotease can be well inhibited, and the expression quantity of the recombinant protein is increased.

Description

Method for improving expression quantity of methanol nutritional yeast expression system

Technical Field

The invention relates to the field of microbial fermentation, in particular to a method capable of improving expression quantity of recombinant protein expressed by a methanol nutritional yeast expression system.

Background

The yeast expression system is one of the most widely applied exogenous gene expression systems at present, and the methanol nutritional yeast has low nutritional requirement, quick growth, cheap culture medium and easy operation and culture; the expressed exogenous protein can be secreted to the outside of cells, the secreted endogenous protein is less, the separation and purification of the exogenous protein are simple and convenient, and particularly, the pichia pastoris in the methanol nutritional yeast is widely applied to production.

However, in the process of expressing recombinant protein, the easily degradable protein is easily degraded by the self-generated enzyme system in the fermentation process, which is very unfavorable for the expression and accumulation of the recombinant protein. Generally, the nitrogen source level in the fermentation process is improved to reduce degradation of the recombinant protein by thallus self protease, the method easily causes material waste and further increases the fermentation cost, the increased nitrogen source does not necessarily have a good effect of improving expression of the recombinant protein, and meanwhile, the residual nitrogen source after fermentation is finished may cause certain difficulty in later-stage extraction and purification. There are also schemes for adding protease inhibitors to the culture medium to inhibit degradation of recombinant proteins, but the effect is not significant, and there is no good method for greatly reducing degradation of recombinant proteins at present, so it is very important to find a method for inhibiting degradation of proteins.

Disclosure of Invention

The invention aims to develop a method for improving the expression quantity of recombinant protein expressed by a methanol nutritional yeast expression system aiming at the defects of the prior art.

In order to achieve the aim, the invention provides a method for improving the expression quantity of a methanol nutritional yeast expression system, which comprises the following steps: in the induction stage of the fermentation of the methylotrophic yeast, the fed-in methanol contains a protease inhibitor.

In some embodiments, the protease inhibitor is benzamidine hydrochloride or phenylmethylsulfonyl fluoride.

In other embodiments, the protease inhibitor is benzamidine hydrochloride.

In some embodiments, the protease inhibitor is present in an amount of 0.005mol/L to 0.15mol/L.

In some embodiments, the methylotrophic yeast is pichia pastoris.

In some embodiments, the expressed recombinant protein is selected from the group consisting of carboxypeptidase B precursor, insulin precursor analog, glucagon-like precursor, or trypsin precursor.

The term "precursor" as used herein refers to a single chain polypeptide from which a protein of interest can be obtained by one or more subsequent chemical and/or enzymatic steps. An "insulin precursor analog" refers to an insulin precursor molecule having one or more mutations, substitutions, deletions, and/or additions of the a and/or B chains relative to the human insulin molecule. Similarly, for example, recombinant expression of glucagon, insulin glargine, insulin aspart, deglutition insulin, insulin lispro, trypsin, carboxypeptidase, etc. by genetic engineering all express their single-chain polypeptides, i.e., precursor proteins, which are well known in the art.

All ranges cited herein are inclusive, unless expressly stated to the contrary. For example, "the content of the protease inhibitor is 0.005mol/L to 0.15mol/L" means that the content of the protease inhibitor n is 0.005 mol/L.ltoreq.nltoreq.0.15 mol/L.

The invention has the advantages that: 1) The content of the added protease inhibitor is less, the cost is low, and the subsequent separation and purification are not influenced; 2) Compared with the direct addition of a fermentation substrate, the protease inhibitor is introduced into the fermentation system along with the methanol feeding, so that the influence on the cell growth is milder, and the recombinant protein expression quantity is further increased.

Detailed Description

The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that various changes and modifications based on the inventive concept herein will occur to those skilled in the art and are intended to be included within the scope of the present invention.

The materials used in the examples are commercially available, the methods of constructing the strains used are well known to those skilled in the art, and the host cells and Expression vectors of the strains used in the present invention are described in reference to Multi-Copy Pichia Expression Kit and easy select from Invitrogen ^TM The Kit description of the Pichia Expression Kit belongs to the technical public knowledge. The Fermentation method of the embodiment of the invention is carried out in Pichia Fermentation Process Guidelines of Invitrogen company, and the concrete operation is as follows:

the fermentation medium consisted of BSM,1% Yeast Extract (Yeast Extract) and PTM 1. Sterilizing BSM and yeast extract at 121 deg.C for 30min, adjusting pH to 5.0 with 50% ammonia water, and adding sterile-filtered PTM1 at a concentration of 4 mL/L.

The fermentation is divided into three steps:

the first step is as follows: culturing the fermentation medium for about 20 hours;

the second step is that: 50% glycerol containing 12mL/L PTM1 was fed in at a rate of 10mL/h/L for 6h.

The third step: the induction was carried out by feeding methanol containing 12mL/L PTM1 at a rate of 6 mL/h/L. In order to enable the DO value in the induction process to be not lower than 25%, the methanol feeding rate needs to be adjusted, the induction is carried out for 72 hours, and the culture is finished after the OD600 of the bacterial liquid reaches more than 500. Taking fermentation liquor in the fermentation culture process, centrifuging, and taking supernatant liquid to detect the protein content by HPLC.

Example 1

Pichia pastoris GS115 recombinant expression carboxypeptidase B precursor

Methanol is fed and induced to express without adding any protease inhibitor, fermentation liquor of 80h and 110h is taken for centrifugation, HPLC is used for measuring the expression quantity of the carboxypeptidase B precursor, and the concentration of 80h is taken as a standard value 100 to be used as a reference. The fermentation was carried out in the same manner before the fermentation induction in the experimental group, 0.05mol/L benzamidine hydrochloride and 0.05mol/L phenylmethylsulfonyl fluoride (PMSF) were added to methanol during the methanol induction, and samples were centrifuged at 80h and 110h, and the expression level of carboxypeptidase B precursor was determined by HPLC, which is shown in Table 1 in comparison with the control experiment.

TABLE 1 Effect of protease inhibitor addition on the amount of carboxypeptidase B precursor expression

Example 2

Pichia pastoris X33 recombinant expression protease precursor

Methanol is fed and induced to express without adding any protease inhibitor, fermentation liquor of 80h and 110h is taken for centrifugation, the expression quantity of trypsin precursor is measured by HPLC, and the concentration of 80h is taken as a standard value 100 as a reference. The fermentation was carried out in the same manner before the induction of the fermentation in the experimental group, and in the course of the induction of the methanol, 0.05mol/L benzamidine hydrochloride and 0.05mol/L phenylmethylsulfonyl fluoride (PMSF) were added to the methanol, and at the same time, 80h and 110h sampling and centrifugation were carried out to measure the expression level of the trypsin precursor by HPLC, which is shown in Table 2 in comparison with the control experiment.

TABLE 2 Effect of protease inhibitor addition on Trypsin precursor expression

Example 3

Pichia pastoris GS115 recombinant expression carboxypeptidase B precursor

Methanol is fed and induced to express without adding any protease inhibitor, fermentation liquor of 80h and 110h is taken for centrifugation, HPLC is used for measuring the expression quantity of the carboxypeptidase B precursor, and the concentration of 80h is taken as a standard value 100 to be used as a reference. The fermentation was performed in the same manner before the fermentation induction in the experimental group, in which 0.001mol/L, 0.005mol/L, 0.05mol/L, 0.10mol/L, 0.15mol/L, 0.20mol/L benzamidine hydrochloride and 0.005mol/L, 0.05mol/L, 0.10mol/L, 0.15mol/L, 0.20mol/L PMSF were added to methanol, respectively, and the samples were centrifuged at 80h and 110h to determine the expression level of carboxypeptidase B precursor, which is shown in the following Table 3 and Table 4 compared with the experimental results in the control group.

TABLE 3 Effect of benzamidine hydrochloride addition on the amount of carboxypeptidase B precursor expressed

TABLE 4 Effect of the addition of phenylmethylsulfonyl fluoride on the amount of expression of the carboxypeptidase B precursor

Example 4

Effect of protease inhibitor addition mode on Pichia pastoris GS115 recombinant expression of carboxypeptidase B precursor

And (3) performing induced expression by methanol feeding, respectively adding benzamidine hydrochloride or PMSF which is equal to the methanol feeding amount to the base material, not adding benzamidine hydrochloride or PMSF to the methanol, centrifuging fermentation liquor for 80h and 110h, measuring the expression amount of a carboxypeptidase B precursor by HPLC, and respectively taking the concentration of 80h as a standard value 100 as a reference. The experimental group was fermented in the same manner before induction of fermentation, the base material was not added with benzamidine hydrochloride or PMSF, the methanol induction process was performed by adding benzamidine hydrochloride of 0.05mol/L concentration and PMSF of 0.05mol/L concentration to methanol, and sampling and centrifuging for 80h and 110h to measure the expression level of carboxypeptidase B precursor by HPLC, which is shown in the following Table 5 in comparison with the control group.

TABLE 5 Effect of the addition of phenylmethylsulfonyl fluoride on the amount of expression of carboxypeptidase B precursor

Claims

1. A method for improving expression quantity of a methylotrophic yeast expression system is characterized in that in a fermentation induction stage, fed-in methanol contains a protease inhibitor, wherein the protease inhibitor is benzamidine hydrochloride, and the content of the protease inhibitor is 0.05mol/L; the methylotrophic yeast is Pichia pastoris.

2. The method of claim 1, wherein the protein is selected from the group consisting of carboxypeptidase B precursor, insulin precursor analog, glucagon-like precursor, and trypsin precursor.