Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase.
The primary structure of several chloroplast fructose-1,6-bisphosphatase (CFBPase) was deduced from DNA sequences, but only spinach, pea and rapeseed enzymes have been characterized structurally. We analyzed whether CFBPases from different phylogenetic origin contain a common epitope. 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-transferase (GT) of Schistosoma japonicun (26.5 kDa). Upon transformation of Escherichia coli and induction with isopropyl-beta-D-thiogalactopyranoside, centrifugation of the lysate partitioned 10% of the fusion protein in the supernatant fraction and the remaining 90% in the precipitate. The expected 65 kDa protein was purified from both the soluble and the particulate fraction by affinity chromatography on columns of glutathione-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 the 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 epitope through biological evolution.
Hagelin
K
K
Instituto de Investigaciones Bioquimicas Luis F. Leloir Fundación Campomar, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Rodriguez-Suarez
R
R
Katzen
F
F
Wolosiuk
R A
RA
Baldi
P C
PC
Giambartolomei
G H
GH
Fossati
C A
CA
Dyer
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D003429:Cross Reactions; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D006597:Fructose-Bisphosphatase / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D005982:Glutathione Transferase / Q000235:genetics; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D010944:Plants / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000201:enzymology / Q000235:genetics; D013045:Species Specificity; D014908:Triticum / Q000201:enzymology* / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D006597:Fructose-Bisphosphatase