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| Expression and Characterization of Fusion Protein of Single-chain Variable Fragment of Alpha Fetoprotein and Allophycocyanin Alpha Subunit |
| CHEN Hua-xin1,2, WU Jing1,2,3, ZHAO Jin1,2, JIANG Peng1,2 |
1. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China;
2. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China;
3. University of Chinese Academy of Sciences, Beijing 100049, China |
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Abstract Objective:By using multiple gene expressing plasmids, fusion protein of single-chain variable fragment of alpha fetoprotein and allophycocyanin alpha subunit, phycobiliprotein lyase (cpcS), and enzymes (Ho1 and pebS) for conversion of heme to phycoerythrobilin were co-expressed in E. coli, with an aim to prepare fluorescent fusion protein which is covalently bound with phycoerythrobilin. Method:fragments of alpha fetoprotein and allophycocyanin alpha subunit were fused by fusion PCR. Then the fused gene, together with cpcS gene, were ligated into vector pCDFDuet-1. Ho1 and pebS genes were ligated into another vector pRSFDuet-1. The two vectors were then transformed into E. coli. Recombinant proteinwere expressed efficiently in E. coli by addition of IPTG and purified by affinity chromatography.The activities of the recombinant protein were evaluated by spectral analysis and competitive ELISA.Results:The recombinant protein, with a molecular weight approximately to 45.0kDa, has an absorbance maximum at 549.5nm and an emission maximum at 660nm. Competitive ELISA results showed that inhibition rate reached 48% when the concentration ratio of scFv and parent monoclonal antibodies was 16:1.Conclusion:A recombinant protein, which was fluorescent and immunologic active, was successfully expressed in E. coli.
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Received: 09 November 2015
Published: 26 January 2016
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