Production and characterization of polyclonal antibodies in rabbits to 4S-limonene synthase from spearmint (Mentha spicata)

Abstract

Limonene synthase, a monoterpene cyclase from the oil glands of spearmint (Mentha spicata) leaves that catalyzes the conversion of geranyl pyrophosphate to (-)-4S-limonene, was purified, and polyclonal antibodies were generated in rabbits against the sodium dodecyl sulfate-denatured protein. Immunoblotting analysis revealed that the antibodies were very specific for denatured limonene synthase from all Mentha species tested. However, no immunological cross-reactivity was observed with denatured limonene synthases from Valencia oranges (Citrus sinensis, Rutaceae) or wormseed (Chenopodium ambrosioides, Chenopodiaceae). Furthermore, the antibody preparation did not detectably cross-react with other monoterpene cyclases from related angiosperm species of the Lamiaceae, Asteraceae, and Umbellifereae, or from conifer species, and no cross-reactivity was demonstrated toward several sesquiterpene cyclases of higher plant and fungal origin. Although the antibody preparation was highly selective for denatured limonene cyclase from Mentha, the antibodies did not recognize the native protein in several different types of experiments. Nevertheless, specificity for the target enzyme was unambiguously demonstrated when the antibody preparation was shown to cross-react with the cyclase protein expressed in Escherichia coli that harbored the corresponding limonene synthase cDNA gene from M. spicata.