Expression of human interleukin-11 cDNA in E. coli.

J Miao, J Wang, S Peng, P Tang, M Zou, J Duan, C Zhao, X Ma
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Abstract

A 551-bp hIL-11 gene fragment that includes no nucleotide sequences encoding signal polypeptide and the initial 8 amino acids of the mature protein was cloned into a high-level expression vector pEx31B of E. coli. The authors identified the recombinant plasmid, designated pEx31-IL11, by restriction endonucleases digestion and DNA sequencing. The resulting recombinant plasmids were then used to transform E. coli strain HB101, and expression in the PL promoter system, which is temperature-regulated, was achieved. The expressed fusion protein amounts to 50% of total bacterial proteins. The hIL-11 protein expressed in E. coli was fused to the N-terminal 99 amino acids of the MS2 polymerase to form the inclusion body. These recombinant proteins can be purified to about 80% by extracting inclusion body with urea. One IL-6-dependent cell line 7 TD1 was used for bioassay. The recombinant hIL-11 protein was preliminarily purified and renatured to a specific activity of 10(5)U/mg, even in the presence of an excess of a neutralizing anti-IL-6 antibody.

人白细胞介素-11 cDNA在大肠杆菌中的表达。
将一个551 bp的hIL-11基因片段克隆到大肠杆菌的高水平表达载体pEx31B中,该片段不含编码信号多肽的核苷酸序列和成熟蛋白的前8个氨基酸。作者通过限制性内切酶和DNA测序鉴定了重组质粒,命名为pEx31-IL11。将重组质粒转化大肠杆菌HB101,在温度调节的PL启动子系统中实现表达。表达的融合蛋白占细菌总蛋白的50%。在大肠杆菌中表达的hIL-11蛋白与MS2聚合酶的n端99个氨基酸融合形成包涵体。用尿素提取包涵体可使重组蛋白纯度达到80%左右。使用il -6依赖性细胞系7td1进行生物测定。重组il -11蛋白被初步纯化并还原为10(5)U/mg的特异性活性,即使存在过量的中和性抗il -6抗体。
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