Thermal stabilization of a single‐chain Fv antibody fragment by introduction of a disulphide bond

FEBS Letters

Young, N.Martin¹  Oomen, Raymond P.¹  MacKenzie, C.Roger¹  Baenziger, John E.²  Narang, Saran A.¹ 

[1] Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ont. K1A 0R6, Canada;Department of Biochemistry, University of Ottawa, Ottawa, Ont. K1H 8M5, Canada

关键词: Antibody engineering;    Fourier transform IR spectroscopy;    Single-chain Fv;    Thermal stability;    EIA;    enzyme immunoassay;    FTIR;    Fourier transform IR spectroscopy;    scFv and ds-scFv;    single-chain Fv and disulphidestabilized scFv;   

DOI  :  10.1016/0014-5793(95)01325-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A disulphide bond was introduced into a single-chain Fv form of the anticarbohydrate antibody, Se155-4 by replacing Ala-L57 of the light chain and Asp-H106 of the heavy chain with cysteines, by site-directed mutagenesis. To maintain the saltbridge from the latter residue to Arg-H98, Tyr-107 was also altered to Asp. The resulting ds-scFv was shown to retain full antigen-binding activity, by enzyme immunoassay and surface plasmon resonance analysis of binding kinetics. Compared with the parent scFv, the disulphide bonded form was shown to have enhanced thermal stability, by Fourier transform IR spectroscopy. The T _m was raised from 6°C to 69°C. The ds-scFv form thus combines the stable monomeric form of the disulphide form with the expression advantages of the scFv.

【 授权许可】

Unknown