Production and characterisation of monoclonal antibodies to phytoene synthase of lycopersicon esculentum

Abstract

Monoclonal antibodies have been prepared against the tomato ( Lycopersicon esculentum Mill.) fruit ripening-enhanced phytoene synthase (PSY1). The antigen was prepared as a fn2 fn2 d-thiogalactopyranoside; mab = monoclonal antibody; PMSF = phenylmethylsulphonyl fluoride; PVDF = polyvinylidene difluoride; TBST = Tris-buffered saline\\Tween; TBS = Tris-buffered saline. -galactosidase fusion protein by cloning a 1.13 kb fragment of Psy1 cDNA into pUR291, followed by transformation of E. coli. The fusion protein, induced by IPTG, was purified by preparative SDS-PAGE and used to elicit an immune response. The cell lines were screened for cross-reactivity against β-galactosidase- phytoene synthase fusion protein in E. coli extracts using western blotting and ELISA detection procedures. Positive clones were further 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. Nine of these, all of the IgM isotype, exhibited strong responses to phytoene synthase of ripe tomato fruit on western blots, but did not inhibit enzyme activity effectively. The other two 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. In contrast, antibodies were unable to detect microbial phytoene synthases, nor phytoene synthase of maize leaf, tomato chloroplast or mango fruit extracts, 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 three different proteins (44, 41, and 37 kDa) in carrot root. Cross-reactivity was also found with a 37 kDa protein from pumpkin fruit stroma.