Rapid isolation of monoclonal antibodies. Monitoring enzymes in the phytochelatin synthesis pathway

Genomics projects have identified thousands of interesting new genes whose protein products need to be examined at the tissue, subcellular, and molecu lar levels. Furthermore, modern metabolic engineering requires accurate con trol of expression levels of multiple enzymes in complex pathways. The lack of specific immune reagents for characterization and monitoring of these n umerous proteins limits all proteomic and metabolic engineering projects. W e describe a rapid method of isolating monoclonal antibodies that required only sequence information from GenBank. We show that large synthetic peptid es were highly immunogenic in mice and crude protein extracts were effectiv e 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 imm unosorbent 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-glut amyl cysteine synthetase, glutathione synthetase, and phytochelatin synthas e. Moreover, immunofluorescence localization studies with antibacterial gam ma -glutamyl cysteine synthetase and antiglutathione synthetase antibodies demonstrated that these immune reagents reacted strongly with their respect ive 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 funct ional studies.