A novel self-cleavable tag Zbasic-∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli.
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 EC50was 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.
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Siwei
S
Shi
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Huanhuan
H
Chen
Jecho Laboratories, Inc., Frederick, MD, USA.
Hua
H
Jiang
Jecho Laboratories, Inc., Frederick, MD, USA.
Yueqing
Y
Xie
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Lei
L
Zhang
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Ninghuan
N
Li
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Chencen
C
Zhu
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Junsheng
J
Chen
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Han
H
Luo
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
Jiaxian
J
Wang
Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai, China.
Lei
L
Feng
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China. roadeer@sjtu.edu.cn.
Huili
http://orcid.org/0000-0002-8748-7899
H
Lu
Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China. jianweiz@sjtu.edu.cn.|Jecho Laboratories, Inc., Frederick, MD, USA. jianweiz@sjtu.edu.cn.
Jianwei
J
Zhu
D001702:Biopharmaceutics / Q000379:methods; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047668:Inteins; D019409:Interleukin-15 / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D013058:Mass Spectrometry; D009169:Mycobacterium tuberculosis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000191:economics / Q000302:isolation & purification; D012995:Solubility
C569274:IL15 protein, human; D019409:Interleukin-15; D011993:Recombinant Fusion Proteins
C-terminal cleavage; Intein; Interleukin-15; Soluble expression; Zbasic