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Thermal stabilization of a single‐chain Fv antibody fragment by introduction of a disulphide bond
Author(s) -
Young N.Martin,
MacKenzie C.Roger,
Narang Saran A.,
Oomen Raymond P.,
Baenziger John E.
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)01325-3
Subject(s) - chemistry , monomer , surface plasmon resonance , mutagenesis , kinetics , immunoglobulin light chain , stereochemistry , residue (chemistry) , thermal stability , site directed mutagenesis , antibody , biochemistry , mutation , organic chemistry , nanoparticle , materials science , physics , quantum mechanics , biology , immunology , gene , nanotechnology , polymer , mutant
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.