The Research on the Search for Biologically Active Natural Products Utilizing Sterol Biosynthetic Enzymes as the Target

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(1) The sequence covering the whole area of human lanosterol synthase was obtained in the previous year. Based on this sequence, the oligo NDAs corresponding to amino and carboxyl termini were synthesized and PCR was carried out to get full length clones. However, the expected product was not obtained. To get a full length clone, two overlapping fragments which had common restriction enzyme recognition site (SacI) in overlapping region, were amplified by PCR and combined after SacI digestion. The obtained full length fragment was ligated to multi-cloning site of yeast expression vector pYES2 in proper direction. The Erg7 deficient mutant of yeast was transformed by the resultant expression plasmid, and the functional complementation by the obtained clone was observed. And lanosterol synthase activity was detected in vitro using tritium labeled oxidosqualene as a substrate in cell free extract of transformed yeast. These results vigorously proved that the obtained clone is the gene encoding human lanosterol synthase. (2) In the similar manner as described above, pea cycloartenol synthase cDNA was also transferred into yeast expression vector and successfully expressed in the wild type yeast. (3) As for the screening of inhibitors from natural products, mutant yeast transformants with cDNAs of human and fungal (not yet cloned) lanosterol synthase were prepared. Simple comparison of growth rates of these transformants with test samples would indicate specific inhibitory activities of natural products contained in the samples. These specific inhibitors would serve as the leads for developping clinical druggs for hypercholestelemia and deep mycosis respectively.