title: Refolded scFv antibody fragment against myoglobin shows rapid reaction kinetics.
abstract: 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 V(H)-V(L) 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 (k(on)/k(off)), 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.
authors.affiliation: Medical Proteomics Research Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-806, Korea. hotdog707@hanmail.net.
authors.forename: Hyung-Nam
authors.initials: HN
authors.lastname: Song
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong 339-700, Korea. jangjunhyuck@naver.com.
authors.forename: Jun-Hyuck
authors.initials: JH
authors.lastname: Jang
authors.affiliation: Department of Food and Biotechnology, Korea University, Sejong 339-700, Korea. ywankim@korea.ac.kr.
authors.forename: Young-Wan
authors.initials: YW
authors.lastname: Kim
authors.affiliation: Department of Biotechnology and Bioinformatics, Korea University, Sejong 339-700, Korea. kdh5611@hanmail.net.
authors.forename: Dong-Hyung
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Medical Proteomics Research Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-806, Korea. sgpark@kribb.re.kr.
authors.forename: Sung-Goo
authors.initials: SG
authors.lastname: Park
authors.affiliation: Medical Proteomics Research Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-806, Korea. mklee@kribb.re.kr.
authors.forename: Myung Kyu
authors.initials: MK
authors.lastname: Lee
authors.affiliation: Department of Biotechnology and Bioinformatics, Korea University, Sejong 339-700, Korea. shpaek@korea.ac.kr.
authors.forename: Se-Hwan
authors.initials: SH
authors.lastname: Paek
authors.affiliation: Medical Proteomics Research Center, Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-806, Korea. ejwoo@kribb.re.kr.
authors.forename: Eui-Jeon
authors.initials: EJ
authors.lastname: Woo
mesh_terms: D006801:Humans; D007700:Kinetics; D009211:Myoglobin / Q000276:immunology*; D058849:Protein Refolding; D057127:Single-Chain Antibodies / Q000737:chemistry*
chemical_list: D009211:Myoglobin; D057127:Single-Chain Antibodies