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首页> 外文期刊>Protein Expression and Purification >Purification and refolding optimization of recombinant bovine enterokinase light chain overexpressed in Escherichia coli
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Purification and refolding optimization of recombinant bovine enterokinase light chain overexpressed in Escherichia coli

机译：在大肠杆菌中过表达的重组牛肠激酶轻链的纯化和重折叠优化

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摘要

The nucleotide sequence encoding bovine enterokinase light chain (EK) from Chinese northern yellow bovine was isolated. Two single-nucleotide mutations, namely, C245G and A528T were identified. The gene encoding the Pro82Arg/Glu176Asp variant of known bovine EK was fused with glutathione S-transferase and overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with IPTG and glucose. Effective fusion protein purification, refolding, auto-catalytic cleavage and mature EK recovery were described. The specific activity of the purified EK was determined as 1 10 +/- 10 U/mg, which was comparable to a specific activity of >= 20 U/mg of the E. coli expressed EK sample provided by Sigma (Cat. No. E4906). This procedure produced approximately 53 mg of EK per 500 mL of cell culture, which was much higher than previous reports, thus providing a basis for large-scale production of EK and for further applications in biotechnology. (c) 2007 Elsevier Inc. All rights reserved.
机译：从中国北方黄牛中分离出编码牛肠激酶轻链（EK）的核苷酸序列。鉴定出两个单核苷酸突变，即C245G和A528T。在用IPTG和葡萄糖诱导后，将已知牛EK的Pro82Arg / Glu176Asp变体的编码基因与谷胱甘肽S-转移酶融合，并主要作为包涵体在大肠杆菌BL21（DE3）中过表达。描述了有效的融合蛋白纯化，重折叠，自动催化裂解和成熟的EK回收。纯化的EK的比活性确定为1 10 +/- 10 U / mg，相当于Sigma提供的大肠杆菌表达的EK样品的比活性> = 20 U / mg。 E4906）。此程序每500 mL细胞培养物中产生约53 mg EK，这比以前的报道要高得多，因此为大规模生产EK和在生物技术中的进一步应用提供了基础。 （c）2007 Elsevier Inc.保留所有权利。

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