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| Construction,Expression,Purification and Site-specific PEGylation of Integrated Interferon Mutant Cys72 |
| HUANG Xiang-lu1,TAO Shan-shan1,XU Chen2,LI Jie2,ZHOU Jian-ping1 |
1.Pharmaceutical Science Department，China Pharmaceutical University，Nanjing 210009，China
2.Beijing Tri-prime Genetic Engineering Co.,Ltd.，Beijing 102600，China |
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Abstract Interferon alpha are used in clinic to treat a variety of viral diseases and cancers. They have short circulating half-life, which necessitates frequent administration to patients. Previous studies showed that it is possible to extend the circulating half-life of interferon alpha by modifying cysteine residues of the protein with poly(ethylene glycol)(PEG) reagents. But protein seldom have unpaired –SH, so a free cysteine residue was introduced into the protein by recombinant DNA technique and only 1% activity was retained after Pegylation. So a proper position should be selected for Pegylation and enhance the retained activity. Integrated Interferon (IIFN) has a wide variety of biological effects that include viral inhibition, antiproliferation, and immunomodulation . Aspartate residue at positon of 72 was chosed by homology sequence analysisi and space structure simulation methods. Integrated interferon mutant Cys72(IIFN72C) was a cysteine mutant of IIFN. The protein had higher specific activity and may used for site-directed modified by PEG. It was constructed by substitution of Asp at position 72 with Cys using site-directed mutagenesis. The DNA was constructed in pET-23b expression vector, and transformed into E.coli BL21(DE3). IIFN72C was expressed as inclusion bodies with yield of more than 30% of total bacterial protein. The recombinant protein was expressed by auto-induction and purified by DEAE Sepharose FF and Chelating Sepharose FF column chromatography. After purification, the protein was modified with a 20kDa mPEG-maleimide and the mono-PEGylated protein was separated from unmodified IIFN72C by stepwise elution. After purification, the purity of mono-PEG-IIFN72C was up to 98%, and the biological activity was more than 3×107IU/mg, The retained activity was up to 8%, which higher than before. These results confirmed the utility of site-specific PEGylation for creating long-acting interferon with higher activity.
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Received: 10 February 2010
Published: 29 April 2010
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