Purification and preparation of antibody scFv TU20 p 643 and p 745 for crystallization and determination structure by x-ray.

B. Uhnáková^*, J. Brynda, M. Fábry, M. Hořejší, V. Král, I.Sieglová, J. Sedláček

Department of Recombinant Expression and Structural Biology, Institute of Molecular Genetics, Flemingovo nám. 2, CZ-166 37 Praha 6

The class III beta-tubulin isotype is widely used as a neuronal marker in normal and neoplastic tissues [1]. This isotype was, however, also immunodetected in certain tumours of non-neuronal origin such as squamous cell carcinoma. The distribution of class III beta-tubulin in normal and neoplastic tissues was compared using a newly described monoclonal antibody. The TU-20 mouse monoclonal antibody was prepared against a conserved synthetic peptide from the C-terminus of the human class III beta-tubulin isotype. Its specificity was confirmed by immunoblotting and by immunofluorescence microscopy on cultured cells. The results indicate a specific TU-20 epitope expression exclusively in neuronal tissues. The antibody could thus be a useful tool for the probing of class III beta-tubulin functions in neurons as well as immunohistochemical characterisation.

Fv fragments are the smallest antibody molecules that still retain the entire antigen-binding site. In single chain Fv fragments (scFv), variable domains are joined by a flexible linker. We have prepared scFv TU20 construct and screened its activity (ELISA). Our objective has been to study the activity and continue to structural studies with improvement in the protein engineering. Two constructs, scFv TU20 p643 and p745 (contains Tyrosine rich domain) have been cultivated and purified for crystallization.

1. E. Dráberová, Z. Lukáš, D. Iványi, V. Viklický, P. Dráber. Histochem Cell Biol. 109, (1998), 231-9.