The combined effect of the gene copy number and chaperone overexpression on the recombinant bovine chymosin production in Pichia pastoris, with mutant ADH2 promoter

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-11-29 DOI:10.1016/j.pep.2024.106636

Fatma Ersöz , Mehmet İnan

{"title":"The combined effect of the gene copy number and chaperone overexpression on the recombinant bovine chymosin production in Pichia pastoris, with mutant ADH2 promoter","authors":"Fatma Ersöz , Mehmet İnan","doi":"10.1016/j.pep.2024.106636","DOIUrl":null,"url":null,"abstract":"<div><div>Chymosin is an enzyme used to coagulate milk, in the cheese industry. This study aimed to increase recombinant production of the chymosin in <em>Pichia pastoris</em> by determining the optimum copy number and overproduction of a Protein Disulfide Isomerase (<em>PpPDI)</em> chaperon protein. <em>Bos taurus</em> chymosin was expressed under the control of a mutant <em>ADH2</em> promoter. The clones containing 1–4 gene copy numbers of the chymosin were constructed using the <em>in vitro</em> cloning method, and the effect of chaperone protein on chymosin secretion was investigated.</div><div>The enzyme production levels are 4, 6.3, 4.5, and 3 IMCU/mL for 1, 2, 3, and 4-copy clones. The secreted chymosin levels increased up to two copies, and increasing the number of copies decreased the secretion level. Therefore, <em>PpPDI</em> was over-expressed in the clones regulated with the <em>ADH2</em> promoter. The over-expression of <em>PDI</em> gene increased chymosin secretion in clones compared to the counterpart host. However, the highest chymosin level was obtained with C2 (2-copy chymosin containing clone; 6.3 IMCU/mL) and C2P2 (2-copy chymosin/2-copy PDI containing clone; 8.2 IMCU/mL).</div><div>The maximum production was 39 IMCU/mL with the clone C2P2 in the fermenter scale production. The enzyme activity increased approximately 2-fold by adding two copies of the chaperone protein. The combined effect of gene copy number and chaperone overexpression on chymosin production was investigated. Two copies of the chymosin and <em>PpPDI</em> genes were the optimum among the tested clones.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106636"},"PeriodicalIF":1.4000,"publicationDate":"2024-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein expression and purification","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1046592824002080","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}

引用次数: 0

Abstract

Chymosin is an enzyme used to coagulate milk, in the cheese industry. This study aimed to increase recombinant production of the chymosin in Pichia pastoris by determining the optimum copy number and overproduction of a Protein Disulfide Isomerase (PpPDI) chaperon protein. Bos taurus chymosin was expressed under the control of a mutant ADH2 promoter. The clones containing 1–4 gene copy numbers of the chymosin were constructed using the in vitro cloning method, and the effect of chaperone protein on chymosin secretion was investigated.

The enzyme production levels are 4, 6.3, 4.5, and 3 IMCU/mL for 1, 2, 3, and 4-copy clones. The secreted chymosin levels increased up to two copies, and increasing the number of copies decreased the secretion level. Therefore, PpPDI was over-expressed in the clones regulated with the ADH2 promoter. The over-expression of PDI gene increased chymosin secretion in clones compared to the counterpart host. However, the highest chymosin level was obtained with C2 (2-copy chymosin containing clone; 6.3 IMCU/mL) and C2P2 (2-copy chymosin/2-copy PDI containing clone; 8.2 IMCU/mL).

The maximum production was 39 IMCU/mL with the clone C2P2 in the fermenter scale production. The enzyme activity increased approximately 2-fold by adding two copies of the chaperone protein. The combined effect of gene copy number and chaperone overexpression on chymosin production was investigated. Two copies of the chymosin and PpPDI genes were the optimum among the tested clones.

Abstract Image

查看原文本刊更多论文

基因拷贝数和伴侣蛋白过表达对ADH2启动子突变的毕赤酵母重组牛凝乳酶产生的联合影响。

凝乳酶是一种在奶酪工业中用于凝固牛奶的酶。本研究旨在通过确定蛋白二硫异构酶（PpPDI）伴侣蛋白的最佳拷贝数和过量生产来增加毕赤酵母中凝乳酶的重组生产。牛凝乳酶在ADH2启动子突变体的控制下表达。采用体外克隆的方法构建了含有1 ~ 4个基因拷贝数的乳糜蛋白酶克隆，并研究了蛋白伴侣对乳糜蛋白酶分泌的影响。1、2、3和4拷贝克隆的酶产量分别为4、6.3、4.5和3 IMCU/mL。分泌的凝乳酶增加到2个拷贝，拷贝数的增加使分泌水平降低。因此，PpPDI在ADH2启动子调控的克隆中过表达。与对应宿主相比，PDI基因的过表达增加了克隆的凝乳酶分泌。然而，C2（2拷贝）含凝乳酶克隆的凝乳酶水平最高；6.3 IMCU/mL)和C2P2(2拷贝凝乳酶/2拷贝含PDI的克隆；8.2 IMCU /毫升)。克隆C2P2在发酵罐规模生产中最高产量为39 IMCU/mL。通过添加两个伴侣蛋白的拷贝，酶活性增加了大约2倍。研究了基因拷贝数和伴侣蛋白过表达对凝乳酶产生的共同影响。其中凝乳酶基因和PpPDI基因的两个拷贝最优。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.