High level soluble expression, purification, and characterization of human ciliary neuronotrophic factor in Escherichia coli by single protein production system.
Ciliary neurotrophic factor (CNTF) is characterized as a neuropoietic cytokine for a broad spectrum of neurons, leading to its evaluation in humans suffering from neurodegenerative diseases. Due to its wide range of biological applications, high yield production of soluble biologically active recombinant human CNTF (rhCNTF) in heterologous expression system is demanded. Many attempts had been undertaken to product rhCNTF in Escherichia coli (E. coli), however, the expression level of rhCNTF was low and most of which formed insoluble inclusion bodies. In this study, we described a new and efficient method to express rhCNTF. The human CNTF gene was codon optimized and then expressed by the single protein production (SPP) expression system in E. coli. The results showed that rhCNTF was expressed as a soluble biologically active protein, and upon purification, the final yield was about 250 mg/L in shake flask with a specific neuroprotective activity in Aβ-induced SH-SY5Y cell injury model. Our study might open up a new strategy for large-scale production of functional rhCNTF for clinical applications as well as basic research.
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province, China. Electronic address: wangke@jsinm.org.
Ke
K
Wang
Faculty of Pharmacy, University of Sydney, NSW 2006, Australia.
Fanfan
F
Zhou
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province, China.
Lan
L
Zhu
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province, China.
Xue
X
Zhu
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province, China.
Kai
K
Zhang
Save Sight Institute, University of Sydney, NSW 2000, Australia.
Ling
L
Zhu
D001483:Base Sequence; D020934:Ciliary Neurotrophic Factor / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D019610:Cytoprotection; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D015316:Genetic Therapy; D006801:Humans; D008969:Molecular Sequence Data; D019636:Neurodegenerative Diseases / Q000628:therapy; D010957:Plasmids / Q000235:genetics
D020934:Ciliary Neurotrophic Factor
Codon optimization; Escherichia coli; Single protein production system; rhCNTF