基于启动子和宿主改造的酿酒酵母表达系统优化研究

doi:10.13523/j.cb.20150109

中国生物工程杂志

2015, Vol. 35

Issue (1): 61-66 DOI: 10.13523/j.cb.20150109

技术与方法

基于启动子和宿主改造的酿酒酵母表达系统优化研究

张旭, 王晶晶, 刘建平

复旦大学生命科学学院遗传工程国家重点实验室上海 200433

The Optimization of Saccharomyces cerevisiae Expression System by Mutagenesis of Promoter and Host Strain

ZHANG Xu, WANG Jing-jing, LIU Jian-ping

State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China

全文: PDF(641 KB) HTML

摘要：

生物医药领域中一套高效表达系统对于重组蛋白的生产至关重要。酿酒酵母作为一种食品级真核微生物,具有繁殖迅速、培养简单、遗传操作便捷等特点,是生产重组蛋白较理想的表达系统之一。对实验室已有的pHR酿酒酵母表达系统进行优化。分别通过易错PCR技术和菌株诱变技术对酿酒酵母启动子P_TEF和宿主酿酒酵母Y16进行突变改造,经筛选、鉴定获得表达性能提高的启动子P_TEFV1和酿酒酵母Y16-E14、Y16-E19。随后,利用启动子P_TEFV1构建以Y16-E14为宿主的pHR-N酿酒酵母表达系统,以绿色荧光蛋白和人血清白蛋白为对象,比较表达系统改造前后性能变化。结果显示pHR-N酿酒酵母表达系统无论胞内表达绿色荧光蛋白还是分泌表达人血清白蛋白的能力均较改造前明显提高。pHR-N系统为获得更多具有重要应用价值的重组蛋白提供了有利的工具。

关键词： 酿酒酵母; 表达系统; 启动子; 宿主; 优化改造

Abstract:

An expression system with high efficiency is very important for recombinant proteins production in biopharmaceutical field. Saccharomyces cerevisiae is a food-graded eukayotic organism. The features of short generation time, simple culture condition and well-characterized manipulation techniques make yeast S. cerevisiae an attractive cell factory for production of heterologous protein. Here, for the purpose to improve the efficiency of pHR expression system which was constructed in our lab previously., the promoter of pHR expression vector (P_TEF) and host strain Y16 were modified by the way of error-prone PCR and mutagenesis respectively. After several rounds of screening, a mutant P_TEFv1 with higher efficiency than the mother promoter P_TEF was obtained. Two modified yeast strains Y16-E14 and Y16-E19 were identified with higher productivity of heterologous protein than yeast Y16. Then P_TEFv1 and yeast Y16-E14 were used to construct the novel pHR-N expression system. To evaluate the ability of pHR-N expression system, yeast green fluorescent protein (GFP) and human serum albumin (HSA) were chosed to be expressed intracellularly and extracellularly respectively. The results showed that pHR-N system had higher ability to produce either intracellular GFP or extracellular HSA than pHR system. The pHR-N yeast expression system provides a valuable resource for future application in recombinant protein production.

Key words: Saccharomyces cerevisiae Expression system Promoter Host strain optimization

收稿日期: 2014-10-21 出版日期: 2015-01-25

ZTFLH:

Q815

基金资助:

国家"863" 计划资助项目(2007AA02Z107)

通讯作者: 刘建平 E-mail: jpliu@fudan.edu.cn

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引用本文:

张旭, 王晶晶, 刘建平. 基于启动子和宿主改造的酿酒酵母表达系统优化研究[J]. 中国生物工程杂志, 2015, 35(1): 61-66.

ZHANG Xu, WANG Jing-jing, LIU Jian-ping. The Optimization of Saccharomyces cerevisiae Expression System by Mutagenesis of Promoter and Host Strain. China Biotechnology, 2015, 35(1): 61-66.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150109 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I1/61

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