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Int. J. Mol. Sci. 2014, 15(12), 23658-23671;

Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

Medical Proteomics Research Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-806, Korea
Department of Biotechnology and Bioinformatics, Korea University, Sejong 339-700, Korea
Department of Food and Biotechnology, Korea University, Sejong 339-700, Korea
Department of Bio-analytical Science, University of Science and Technology, Daejeon 305-333, Korea
Authors to whom correspondence should be addressed.
Received: 16 September 2014 / Revised: 1 December 2014 / Accepted: 12 December 2014 / Published: 18 December 2014
(This article belongs to the Collection Protein Folding)
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Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the VHVL sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10−4 M−1·s−1 and 6.29 × 10−3 s−1, respectively, with an affinity value exceeding 107 M−1 (kon/koff), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor. View Full-Text
Keywords: single-chain variable fragment (scFv); premature antibody; myoglobin; acute myocardial infarction single-chain variable fragment (scFv); premature antibody; myoglobin; acute myocardial infarction

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Song, H.-N.; Jang, J.-H.; Kim, Y.-W.; Kim, D.-H.; Park, S.-G.; Lee, M.K.; Paek, S.-H.; Woo, E.-J. Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics. Int. J. Mol. Sci. 2014, 15, 23658-23671.

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