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Plant Physiol, November 2001, Vol. 127, pp. 711-719
Rapid Isolation of Monoclonal Antibodies. Monitoring Enzymes in
the Phytochelatin Synthesis Pathway1
Yujing
Li,
Muthugapatti K.
Kandasamy, and
Richard B.
Meagher*
Department of Genetics, University of Georgia, Athens, Georgia
30602
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  -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.
1
This work was supported by the Department of
Energy's Environmental Management Science Program (grant no.
DEG0796ER20257) and by the National Institutes of Health Molecular
Biology Program (2RO1GM#36397-14).
*
Corresponding author; e-mail meagher{at}arches.uga.edu; fax
706-542-1387.
© 2001 American Society of Plant Physiologists
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