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...
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1996
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01455680_v42_n5_p673_Hagelin http://hdl.handle.net/20.500.12110/paper_01455680_v42_n5_p673_Hagelin |
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paper:paper_01455680_v42_n5_p673_Hagelin2023-06-08T15:12:21Z Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase. Hagelin, Karin Rodriguez Suarez, Roberto J. Wolosiuk, Ricardo Alejandro epitope fructose bisphosphatase glutathione transferase hybrid protein monoclonal antibody peptide fragment amino acid sequence animal antibody specificity article chloroplast comparative study cross reaction enzymology Escherichia coli genetics immunology isolation and purification molecular cloning molecular evolution mouse plant Schistosoma japonicum species difference wheat Amino Acid Sequence Animals Antibodies, Monoclonal Antibody Specificity Chloroplasts Cloning, Molecular Cross Reactions Epitopes Escherichia coli Evolution, Molecular Fructose-Bisphosphatase Glutathione Transferase Mice Peptide Fragments Plants Recombinant Fusion Proteins Schistosoma japonicum Species Specificity Triticum 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. Fil:Hagelin, K. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Rodriguez-Suarez, R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Wolosiuk, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 1996 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01455680_v42_n5_p673_Hagelin http://hdl.handle.net/20.500.12110/paper_01455680_v42_n5_p673_Hagelin |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
epitope fructose bisphosphatase glutathione transferase hybrid protein monoclonal antibody peptide fragment amino acid sequence animal antibody specificity article chloroplast comparative study cross reaction enzymology Escherichia coli genetics immunology isolation and purification molecular cloning molecular evolution mouse plant Schistosoma japonicum species difference wheat Amino Acid Sequence Animals Antibodies, Monoclonal Antibody Specificity Chloroplasts Cloning, Molecular Cross Reactions Epitopes Escherichia coli Evolution, Molecular Fructose-Bisphosphatase Glutathione Transferase Mice Peptide Fragments Plants Recombinant Fusion Proteins Schistosoma japonicum Species Specificity Triticum |
| spellingShingle |
epitope fructose bisphosphatase glutathione transferase hybrid protein monoclonal antibody peptide fragment amino acid sequence animal antibody specificity article chloroplast comparative study cross reaction enzymology Escherichia coli genetics immunology isolation and purification molecular cloning molecular evolution mouse plant Schistosoma japonicum species difference wheat Amino Acid Sequence Animals Antibodies, Monoclonal Antibody Specificity Chloroplasts Cloning, Molecular Cross Reactions Epitopes Escherichia coli Evolution, Molecular Fructose-Bisphosphatase Glutathione Transferase Mice Peptide Fragments Plants Recombinant Fusion Proteins Schistosoma japonicum Species Specificity Triticum Hagelin, Karin Rodriguez Suarez, Roberto J. Wolosiuk, Ricardo Alejandro Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase. |
| topic_facet |
epitope fructose bisphosphatase glutathione transferase hybrid protein monoclonal antibody peptide fragment amino acid sequence animal antibody specificity article chloroplast comparative study cross reaction enzymology Escherichia coli genetics immunology isolation and purification molecular cloning molecular evolution mouse plant Schistosoma japonicum species difference wheat Amino Acid Sequence Animals Antibodies, Monoclonal Antibody Specificity Chloroplasts Cloning, Molecular Cross Reactions Epitopes Escherichia coli Evolution, Molecular Fructose-Bisphosphatase Glutathione Transferase Mice Peptide Fragments Plants Recombinant Fusion Proteins Schistosoma japonicum Species Specificity Triticum |
| description |
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. |
| author |
Hagelin, Karin Rodriguez Suarez, Roberto J. Wolosiuk, Ricardo Alejandro |
| author_facet |
Hagelin, Karin Rodriguez Suarez, Roberto J. Wolosiuk, Ricardo Alejandro |
| author_sort |
Hagelin, Karin |
| title |
Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase. |
| title_short |
Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase. |
| title_full |
Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase. |
| title_fullStr |
Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase. |
| title_full_unstemmed |
Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase. |
| title_sort |
interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase. |
| publishDate |
1996 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01455680_v42_n5_p673_Hagelin http://hdl.handle.net/20.500.12110/paper_01455680_v42_n5_p673_Hagelin |
| work_keys_str_mv |
AT hagelinkarin interspeciescrossreactivityofamonoclonalantibodydirectedagainstwheatchloroplastfructose16bisphosphatase AT rodriguezsuarezrobertoj interspeciescrossreactivityofamonoclonalantibodydirectedagainstwheatchloroplastfructose16bisphosphatase AT wolosiukricardoalejandro interspeciescrossreactivityofamonoclonalantibodydirectedagainstwheatchloroplastfructose16bisphosphatase |
| _version_ |
1768545181905190912 |