鸢尾素在大肠杆菌中的重组表达及纯化
Recombinant expression and purification of Irisin in Escherichia coli
崔铮琦 1饶昕雨 2张戈瑞 2高子杨 2王龙2
作者信息
- 1. 湖北科技学院医学部药学院(湖北咸宁 437100)
- 2. 湖北科技学院医学部基础医学院(湖北咸宁 437100)
- 折叠
摘要
目的 在大肠杆菌中重组表达并纯化得到高产量、高纯度的鸢尾素(Irisin)重组蛋白,为后续Irisin的功能研究奠定基础.方法 对Irisin基因CDS序列密码子优化后进行基因合成,通过一步克隆连接法构建重组质粒pET-30a-Irisin,将其转化到大肠杆菌表达感受态细胞Rosetta(DE3)中.以终浓度为0.5 mM的IPTG在15 ℃、100 rpm诱导表达30 h.诱导表达结束后收集菌体,超声波破碎细菌细胞,离心取上清,用Ni-IDA琼脂糖纯化树脂进行纯化.结果 成功构建得到重组质粒pET-30a-Irisin,重组表达菌株经低温、低浓度IPTG诱导,经Ni-IDA琼脂糖纯化树脂纯化得到产量为105 mg·L-1的高纯度Irisin重组表达产物.结论 密码子和诱导条件优化可促进Irisin重组蛋白在大肠杆菌中的可溶性表达,为研究其对不同肿瘤细胞的影响及机制奠定坚实基础.
Abstract
Objective To obtain high-yield and high-purity recombinant Irisin protein by recombinant expression and purification in Escherichia coli,and to lay a foundation for the subsequent study of Irisin function.Methods The CDS sequence of the Irisin gene was codon optimized for gene synthesis,and the recombinant plasmid pET-30a-Irisin was constructed by a one-step cloning and ligation method,and was transformed into Escherichia coli expression receptor cells Rosetta(DE3).The expression was induced by IPTG at a final concentration of 0.5 mM for 30 h at 15 ℃ and 100 rpm.The bacteria were collected at the end of induced expression,and the bacterial cells were broken by ultrasonic waves,the supernatant was obtained by centrifugation and purified by Ni-IDA agarose purification resin.Results The recombinant plasmid pET-30a-Irisin was successfully constructed,and the recombinant expression strain was induced by low temperature and low concentration of IPTG,and the recombinant expression product of high-purity Irisin with a yield of 105 mg/L was obtained by purification with Ni-IDA agarose purification resin.Conclusion The optimization of codon and induction can promote the soluble expression of Irisin recombinant protein in Escherichia coli,which lays a solid foundation for the study of its effects and mechanism on different tumor cells.
关键词
鸢尾素/大肠杆菌/重组表达/纯化Key words
Irisin/Escherichia coli/Recombinant expression/Purification引用本文复制引用
出版年
2024
NETL
NSTL
万方数据