重组人白细胞介素-2 在大肠杆菌中的可溶性表达及其简便生产。

IF 1.4 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS
Minhui Zhang , Yongxiang Zheng , Sa Wang, Pengyu Wang, Jingbei Huang, Xiaotong Song, Rong Yu, Chun Zhang
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引用次数: 0

摘要

重组人白细胞介素-2(rhIL-2)是最难在大肠杆菌中生产的细胞因子之一,因为它具有极强的疏水性,极易形成包涵体。rhIL-2 包涵体的再折叠总是意味着繁琐的下游过程和较低的生产效率。在此,我们揭示了一种融合策略,可在大肠杆菌 Origami B (DE3) 宿主中高效可溶性表达并方便生产 rhIL-2。我们设计了一种双串联 SUMO 融合伙伴(His-2SUMO),其 C 端具有独特的 SUMO 蛋白酶裂解位点,可与 rhIL-2 的 N 端融合,融合蛋白(His-2SUMO-rhIL-2)几乎完全以可溶性形式表达。融合伙伴可通过 Ulp1 裂解有效去除,而 rhIL-2 则可通过两步 Ni-NTA 亲和层析简单生成,纯度和整体回收率相当高。对最终获得的 rhIL-2 进行了表征,结果表明纯化后的 rhIL-2 结构紧密有序。虽然最终获得的 rhIL-2 以可溶性聚集体形式存在,而且可能是在表达阶段发生了聚集,但可溶性 rhIL-2 聚集体仍然表现出与市售 rhIL-2 药物制剂相当的生物活性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Soluble expression of recombinant human interleukin-2 in Escherichia coli and its facile production

Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation.

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来源期刊
Protein expression and purification
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.
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