Refolded scFv antibody fragment against myoglobin shows rapid reaction kinetics.

Hyung-Nam Song,Young-Wan Kim,Dong-Hyung Kim,Se-Hwan Paek,Myung Lee,Sung-Goo Park,Jun-Hyuck Jang,Eui-Jeon Woo

doi:10.3390/ijms151223658

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 × 10−4 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.

Highlights

A premature antibody with rapid reaction kinetics was found, and its characteristics have been reported [1,2,3,4,5]
The scFv gene was constructed from the monoclonal antibody of two- to seven-days-old hybridoma cell clones immunized using myoglobin antigen (Figure 1A)
The pelB leader sequence, which directs the protein to the bacterial periplasm in ordinary bacterial expression of scFv for correct folding, was removed in this system because the initial expression and solubility was too low to detect (Figure 1B)

Summary

Introduction

A premature antibody with rapid reaction kinetics was found, and its characteristics have been reported [1,2,3,4,5]. Repetitive exposure to an antigen over an extended period of time gradually induces somatic hypermutation to produce ordinary antibodies that recognize their antigen with a slow dissociation rate and high binding affinity (e.g., 1000-fold), whereas the premature antibody is thought to be generated during an early stage of production. This is referred to as affinity maturation, which consists of the ongoing generation of B-cells receptor (BCR) due to somatic hypermutation and selection of these cells on the basis of affinity. IgM is a temporary antibody that disappears within two or three weeks, which is replaced by IgG [6]

Results

Discussion

Conclusion