Monoclonal antibodies used to characterize cDNA clones expressing specific wheat endosperm proteins

Abstract

A wheat cDNA library, prepared from grain endosperm poly A + -mRNA and cloned into the Escherichia coli expression vector lambda gt11, has been screened with nine monoclonal antibodies having specificities for different wheat endosperm proteins. At least one positive cDNA clone was isolated, and purified, from those selected with each antibody. Each purified cDNA clone was induced to express fusion proteins, and the nitrocellulose membranes to which the proteins were transferred were incubated with each of the other antibodies at two or more concentrations to investigate the extent of homologies between expressed fusion proteins. The specificities of the antibodies were determined using immunoblotting under the same conditions used for binding to the fusion proteins from the expressed cDNA clones. Denatured DNA from each antibody-selected cDNA clone was also characterized by hybridization to αa-/β-genomic gliadin and genomic high molecular weight glutenin subunit DNA probes. Northern hybridizations using the isolated cDNAs as probes for endosperm mRNA were also used to assist clone identification. Some monoclonal antibodies with overlapping specificities (identified from blotting and ELISA experiments) cross-reacted with several expressed cDNA clones. However, in other instances, positive identifications were made of the proteins coded for by single families of the cDNA clones. Monoclonal antibody screening of a wheat cDNA library is useful in identifying families of cDNA clones corresponding to different wheat polypeptides at the primary screening stage, obviating the need in many instances for the application of more tedious methods of clone identification.