Protein Engineering, Vol. 12, No. 7, 623-630,
July 1999
© 1999 Oxford University Press
Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs
Department of Biochemistry, Health Science Building, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5 and 2 Plant Biotechnology Institute, National Research Council of Canada,110 Gymnasium Place, Saskatoon, Saskatchewan, S7N 0W9, Canada 1 Present address: Cancer Immunobiology Center, University of Texas, Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas Texas,7234-8576, USA
The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide–heavy chain–linker–light chain–(His)5 tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10–30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with Kd values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 ± 2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage (~6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.
Keywords: antibody/HPr/gene synthesis/protein binding constant/protein folding/single-chain Fv
3 To whom correspondence should be addressed
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Swennen, M.-F. Paul, L. Vernis, J.-M. Beckerich, A. Fournier, and C. Gaillardin Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis Microbiology, January 1, 2002; 148(1): 41 - 50. [Abstract] [Full Text] [PDF]
|
|