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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2013, Vol. 33 Issue (10): 21-27    DOI:
    
Purification and Characterization of a New Recombinant Cilinary Neuronotrophic Factor Mutant Expressed in Soluble Form by E.coli
FENG Cui1, ZHAO Da-wei2, ZHANG Chun3, WANG Jian4, QIN Pei-yong1, LIU Yong-dong2, SU Zhi-guo2
1. College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China;
2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China;
3. West China Shool of Pharmacy, Sichuan University, Chengdu 610041, China;
4. National Vaccine & Serum Institute Co., Ltd, Beijing 100024, China
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Abstract  Ciliary neurotrophic factor (CNTF) can promote the survival and differentiation of motor neurons and is a potential therapeutic for treating neurodegeneration and nerve injury. Recent research finds it also has great potential as a diet reducing aid. In order to develop its site-specific modified long-acting counterpart, a new human CNTF mutant with a free cystine (CNTF-C17) expressed in soluble form by E.coli has been purified and featured. By combining three different chromatographic steps including hydrophobic interaction chromatography, ion exchange chromatography and affinity chromatography, CNTF-C17 was efficiently separated from bacterial contaminants with a purification factor of 11.4 and a recovery of 35.5%. The purity of target protein reached 98% determined by RP-HPLC. Although this new mutant has a free cysteine, it was found to exist only in monomer form, judged from the elution volume in HP-SEC. CD and FL spectra showed correct secondary and tertiary structure of the purified protein. The molecular weight of this new mutant was 21146 Da, consistent with its theoretical value. Its specific bioactivity was 2.1 ×106 U/mg determined by Tf-1 cell activity detection method. All these data and results laid a foundation for the large production and application of this new CNTF mutant.

Key words： Recombinant human ciliary neurotrophic factor      Cystine      Soluble expression      Separation and purification      Combinatorial chromatography     
Received: 25 April 2013      Published: 25 October 2013
ZTFLH:  Q503  
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FENG Cui
ZHAO Da-wei
ZHANG Chun
WANG Jian
QIN Pei-yong
LIU Yong-dong
SU Zhi-guo
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FENG Cui, ZHAO Da-wei, ZHANG Chun, WANG Jian, QIN Pei-yong, LIU Yong-dong, SU Zhi-guo. Purification and Characterization of a New Recombinant Cilinary Neuronotrophic Factor Mutant Expressed in Soluble Form by E.coli. China Biotechnology, 2013, 33(10): 21-27.

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https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2013/V33/I10/21

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