Preparation of a polyclonal antibody against hypericin synthase and localization of the enzyme in red-pigmented Hypericum perforatum L. plantlets.

Abstract

Hypericum perforatum is well known for its antidepressant and anti-inflammatory activities, for which hypericin and its derivatives are indicated to be the most active compounds. Hypericin synthase (Hyp-1) is the only protein proven to catalyze the synthesis of hypericin. In this study, the full-length cDNA of Hyp-1 was chemically synthesized according to the Hyp-1 sequence in GenBank (accession no. AY148090) and then cloned into the plasmid pET22b. Hyp-1 was expressed in Escherichia coli BL21 (DE3) and purified with a Ni-NTA column. The purified protein was used to immunize New Zealand white rabbits, from which an antiserum was purified by protein G affinity chromatography. The polyclonal antibody against Hyp-1 provides a valuable tool for the study of hypericin biosynthesis in H. perforatum. Expression of Hyp-1 and the cellular distribution of hypericin were analyzed in different organs of red-pigmented H. perforatum plantlets. The black glands were not the only site of hypericin accumulation and the results indicated that hypericin might be synthesized in mesophyll cells or in tissues of the root and/or stem and then transported to the glands. This work provides a foundation for further investigation of the regulatory mechanism of hypericin synthesis during the development of H. perforatum.