当前位置： SCI文献检索 > APPLIED MICROBIOLOGY AND BIOTECHNOLOGY期刊下所有文献 > A novel self-cleavable tag Zbasic-∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli.

A novel self-cleavable tag Zbasic-∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli.

Abstract：

:Soluble expression of recombinant therapeutic proteins in Escherichia coli (E. coli) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic-intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable Mycobacterium tuberculosis recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein "Zbasic-∆I-CM-IL-15" was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic-∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC50 was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic-∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in E. coli.

journal_name

Appl Microbiol Biotechnol

journal_title

Applied microbiology and biotechnology

authors

Shi S,Chen H,Jiang H,Xie Y,Zhang L,Li N,Zhu C,Chen J,Luo H,Wang J,Feng L,Lu H,Zhu J

doi

10.1007/s00253-016-7848-2

subject

Has Abstract

pub_date

2017-02-01 00:00:00

pages

1133-1142

issue

3

eissn

0175-7598

issn

1432-0614

pii

10.1007/s00253-016-7848-2

journal_volume

101

pub_type

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