The Production And Characterization Of Monoclonal Antibodies To Aspartate Transcarbamoylase From Escherichia Coli

Abstract

This research project focused on the production of monoclonal antibodies to the holoenzyme aspartate transcarbamoylase (ATCase) and to its component catalytic and regulatory subunits. The hybridoma cell lines were prepared using immunized splenocytes taken from BALB/c mice and a murine non-immunoglobulin secretor myeloma cell line Sp2/0-Ag/4. In addition to monoclonal antibody production this project also initiated the determination of the specific antigenic determinants for each hybridoma cell line. Elucidation of the epitopes utilized ATCase (pyrBI)-𝛼𝛽-Galactosidase (lacZ) gene fusion products. By 31 sequential deletion of portions of pyrBI, various sized resulting polypeptides, each containing a constant 𝛼-complementing lacZ domain, were produced. The fusion sites in the pyrBI bicistron were located using agarose gel electrophoresis and dideoxy nucleotide sequencing. Resulting fusion protein-products were purified using an anti-𝛽-Galactosidase immuno-affinity minicolumn. Once sequenced and purified, the fusion proteins can be tested with each monoclonal antibody sera to determine antigenic specificity.