Thermal stabilization of a single-chain Fv antibody fragment by introduction of a disulphide bond.
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 salt-bridge 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 Tm was raised from 60 degrees C to 69 degrees C. The ds-scFv form thus combines the stable monomeric form of the disulphide form with the expression advantages of the scFv.
Young
N M
NM
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ont.
MacKenzie
C R
CR
Narang
S A
SA
Oomen
R P
RP
Baenziger
J E
JE
D001665:Binding Sites; D004220:Disulfides; D006361:Heating; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D011487:Protein Conformation; D012475:Salmonella / Q000276:immunology
D004220:Disulfides; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv