Production and characterisation of monoclonal antibodies to phytoene synthase of Lycopersicon esculentum.
Fraser P. D., Misawa N., Sandmann G., Johnson J., Schuch W., Bramley P. M.
Author Affiliation: Division of Biochemistry, School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EW, UK.
Phytochemistry 49 : 971-978
Abstract : Monoclonal antibodies were prepared against tomato fruit ripening-enhanced phytoene synthase (PSY1). The antigen was prepared as a ?-galactosidase fusion protein by cloning a 1.13 kb fragment of Psy1 cDNA into pUR291, followed by transformation of Escherichia coli. The fusion protein was purified by preparative SDS-PAGE and used to elicit an immune response. Cell lines were screened for cross-reactivity against ?-galactosidase-phytoene synthase fusion protein in E. coli extracts using Western blotting and ELISA detection procedures. Positive clones were screened for their ability to cross-react with the mature phytoene synthase protein on Western blots as well as their ability to inhibit enzyme activity. Eleven monoclonal lines were obtained; 9, all of the IgM isotype, exhibited strong responses to phytoene synthase of ripe tomato fruits on Western blots, but did not inhibit enzyme activity effectively. The other 2 lines (IgG/1a 2 isotypes) inhibited phytoene synthase activity in ripe tomato stroma, but produced a poor response to the protein on Western blots. The monoclonals identified a ripe fruit phytoene synthase of 38 kDa, exclusively located in the chromoplast. Antibodies were unable to detect microbial phytoene synthases or phytoene synthase of maize leaves, tomato chloroplasts or mango fruits, either on Western blots or from inhibition of phytoene synthase activity. However, they did cross-react with a 44 kDa protein from carrot leaf stroma and with 3 different proteins (44, 41 and 37 kDa) in carrot roots. Cross-reactivity was also found with a 37 kDa protein from pumpkin fruit stroma.