INTERSPECIES CROSS-REACTIVITY OF A MONOCLONAL-ANTIBODY DIRECTED AGAINST WHEAT CHLOROPLAST FRUCTOSE-1,6-BISPHOSPHATASE

The primary structure of several chloroplast fructose-1,6-bisphosphata se (CFBPase) was deduced from DNA sequences, but only spinach, pea and rapeseed enzymes have been characterized structurally. We analyzed wh ether CFBPases from different phylogenetic origin contain a common epi tope. To this end a DNA fragment of 1200 base pairs encoding 338 amino acid residues of wheat CFBPase (38 kDa) was cloned in the expression plasmid pGEX-1 in frame with the gene coding for glutathione S-transfe rase (GT) of Schistosoma japonicun (26.5 kDa). Upon transformation of Escherichia coli and induction with isopropyl-beta-D-thiogalactopyrano side, centrifugation of the lysate partitioned 10% of the fusion prote in in the supernatant fraction and the remaining 90% in the precipitat e. The expected 55 kDa protein was purified from both the soluble and the particulate fraction by affinity chromatography on columns of glut athione-agarose. This fusion protein was successfully used to produce a monoclonal antibody that specifically recognized the CFBPase region of the fusion protein but not the GT moiety. Moreover, the monoclonal antibody immunoreacted not only with polypeptides (ca. 40 kDa) present in leaf crude extracts of other varieties of wheat (Triticum spelta, T. aestivum and T. durum), but also with homogeneous preparations of t he spinach (Spinacia oleracea) and rapeseed (Brassica napus) enzymes. Thus, the cross reaction of this monoclonal antibody with counterparts from different plant species indicates the persistency of a common ep itope through biological evolution.