title: Rapid isolation of monoclonal antibodies. Monitoring enzymes in the phytochelatin synthesis pathway.
abstract: Genomics projects have identified thousands of interesting new genes whose protein products need to be examined at the tissue, subcellular, and molecular levels. Furthermore, modern metabolic engineering requires accurate control of expression levels of multiple enzymes in complex pathways. The lack of specific immune reagents for characterization and monitoring of these numerous proteins limits all proteomic and metabolic engineering projects. We describe a rapid method of isolating monoclonal antibodies that required only sequence information from GenBank. We show that large synthetic peptides were highly immunogenic in mice and crude protein extracts were effective sources of antigen, thus eliminating the time-consuming step of purifying the target proteins for antibody production. A case study was made of the three-enzyme pathway for the synthesis of phytochelatins. Enzyme-linked immunosorbent assays and western blots with the recombinant proteins in crude extracts demonstrated that the monoclonal antibodies produced to synthetic peptides were highly specific for the different target proteins, gamma-glutamyl cysteine synthetase, glutathione synthetase, and phytochelatin synthase. Moreover, immunofluorescence localization studies with antibacterial gamma-glutamyl cysteine synthetase and antiglutathione synthetase antibodies demonstrated that these immune reagents reacted strongly with their respective target proteins in chemically fixed cells from transgenic plants. This approach enables research to progress rapidly from the genomic sequence of poorly characterized target genes, to protein-specific antibodies, to functional studies.
authors.affiliation: Department of Genetics, University of Georgia, Athens, GA 30602, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.forename: M K
authors.initials: MK
authors.lastname: Kandasamy
authors.forename: R B
authors.initials: RB
authors.lastname: Meagher
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005818:Genetic Engineering; D005721:Glutamate-Cysteine Ligase / Q000276:immunology / Q000378:metabolism*; D005978:Glutathione; D005981:Glutathione Synthase / Q000276:immunology / Q000378:metabolism*; D006825:Hybridomas; D007150:Immunohistochemistry; D008667:Metalloproteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D054811:Phytochelatins; D030821:Plants, Genetically Modified; D020543:Proteome / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D008667:Metalloproteins; D019151:Peptide Library; D020543:Proteome; D054811:Phytochelatins; D019881:Aminoacyltransferases; C093784:glutathione gamma-glutamylcysteinyltransferase; D005721:Glutamate-Cysteine Ligase; D005981:Glutathione Synthase; D005978:Glutathione