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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2015, Vol. 35 Issue (5): 15-21    DOI: 10.13523/j.cb.20150503
    
Preparation of mPEG-MAL-modified Recombinant Human Ciliary Neurotrophic Factor and Evaluation of Biological Activity
FENG Cui1, WANG Qi2, ZHANG Chun2, QIN Pei-yong1, ZHANG Xiu-yu2, WANG Jian3, 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. National Vaccine & Serum Institute, Beijing 100024, China
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Abstract  

The site-direct modification conditions for mutant CNTF-C17 with a molecular weight of 40kDa amaleimide-PEG (mPEG-MAL) was optimized. The mono-PEG-CNTF-C17 was obtained through ion-exchange chromatography and characterized by CD and FL. Experimental data showed that the optimal ratio of protein to PEG was 1:3 and reaction condition was at 4℃ for 12h in Tris-HCl buffer. At the optimized condition, the PEGylatoin yield of mono-PEG-CNTF-C17 can easily reach more than 90%. By anion exchange chromatography mPEG40K-MAL-CNTF-C17 could efficiently separated from the modified mixture with a purity of 98% and a recovery of 78.1%. FL and CD spectra illustrated that mono-PEG-CNTF-C17 has almost the same second and tertiary structure as the natural protein. After PEGylation, the specific activity of CNTF-C17 reduced to 6.51 × 105IU/mg measured by TF-1.CN5a.1 cell. But the half-life in vivo was prolonged 30 times. This study provided a foundation for developing new long-acting CNTF product.

Key words： mPEG-MAL      Mono-PEG-CNTF-C17      Recombinant human ciliary neurotrophic factor      Separation and purification     
Received: 11 November 2014      Published: 25 May 2015
ZTFLH:  Q-31  
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Cite this article:

FENG Cui, WANG Qi, ZHANG Chun, QIN Pei-yong, ZHANG Xiu-yu, WANG Jian, LIU Yong-dong, SU Zhi-guo. Preparation of mPEG-MAL-modified Recombinant Human Ciliary Neurotrophic Factor and Evaluation of Biological Activity. China Biotechnology, 2015, 35(5): 15-21.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20150503     OR     https://manu60.magtech.com.cn/biotech/Y2015/V35/I5/15


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