| Autor: |
Shi, Siwei, Chen, Huanhuan, Jiang, Hua, Xie, Yueqing, Zhang, Lei, Li, Ninghuan, Zhu, Chencen, Chen, Junsheng, Luo, Han, Wang, Jiaxian, Feng, Lei, Lu, Huili, Zhu, Jianwei |
| Předmět: |
|
| Zdroj: |
Applied Microbiology & Biotechnology; Feb2017, Vol. 101 Issue 3, p1133-1142, 10p |
| Abstrakt: |
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 EC 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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
|
