bioaffinity Authors
Yoichi Kumada, Tomomi Kawasaki, Yoshinobu Sakan, Yasufumi Kikuchi, Shigeo Katoh
First author
Yoichi Kumada
Corresponding author
Shigeo Katoh
Publication Style
Journal name Journal of Chemical Engineering of Japan
Year
Volume, issue, pages
41(5) 413-419
Abstract
Effects of nucleotide sequences of polypeptide linkers on production of soluble single-chain Fv antibodies in E. coliwere studied using scFv genes, in which genes of VH and VL domains were genetically linked with 4 types of linker genes with or without rare codons, namely genes of scFv (G4S)3, scFv (G4S)3R, scFv No. 10 and scFv No. 10NR. The transformants expressing scFvs having linkers with 3–4 rare codons (scFv (G4S)3R and scFv No. 10) could grow to higher cell concentrations under induction condition, while growth of the cells expressing scFvs having linkers without rare codon (scFv (G4S)3 and scFv No. 10NR) stopped after 3 h. Productivity levels of soluble scFv by cells expressing scFv (G4S)3R and scFv No. 10 were higher than those of scFv (G4S)3 and scFv No. 10NR. The specific productivity levels of inclusion bodies for scFv (G4S)3R and scFv No. 10 decreased in comparison with those for scFv (G4S)3 and scFv No. 10NR. This might show that decrease in the translation rate at the region coding polypeptide linkers with rare codons has a favorable effect for the subsequent folding process of scFv molecules. Furthermore, from a SPR analysis using an antigen-immobilized sensor chip, scFv (G4S)3R showed the same binding affinity as the original scFv (G4S)3. Thus, nucleotide sequences of polypeptide linkers are important to increase productivity of scFv expressed in soluble fraction. The nucleotide sequence of the flexible linker (G4S)3R used in this study may be useful for efficient production of various scFvs without loss of the binding affinity of target scFv.