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Title 

Refolded scFv antibody fragment against myoglobin shows rapid reaction kinetics

Authors 

Hyeong Nam SongJ H JangY W KimD H KimSung Goo ParkMyung Kyu LeeS H PaekEui-Jeon Woo

Publisher 

MDPI AG

Issue Date 

2014

Citation 

International Journal of Molecular Sciences, vol. 15, no. 12, pp. 23658-23671

Keywords 

Acute myocardial infarctionMyoglobinPremature antibodySingle-chain variable fragment (scFv)

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 VH-VL 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?4M?1·s?1and 6.29 × 10?3s?1, respectively, with an affinity value exceeding 107M?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.

ISSN 

1422-0067

Link 

http://dx.doi.org/10.3390/ijms151223658

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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