HIGH-LEVEL EXPRESSION OF MAMMALIAN PROTEIN FARNESYLTRANSFERASE IN A BACULOVIRUS SYSTEM - THE PURIFIED PROTEIN CONTAINS ZINC

The mammalian enzyme protein farnesyltransferase is a heterodimeric pr otein that catalyzes the addition of a farnesyl isoprenoid to a cystei ne in ras proteins. Since oncogenic forms of ras proteins require the farnesyl group for transforming activity, the structure and mechanism of this enzyme are important to define. However, such studies have bee n difficult to approach because of the low abundance of the enzyme in mammalian tissues and hence the problems of obtaining large quantities of the protein. We report here the coexpression of the two subunits o f protein farnesyltransferase by Sf9 cells infected with a recombinant baculovirus containing the coding sequences of both polypeptides. Thi s results in the production of milligram quantities of enzyme which ca n be readily purified by conventional chromatographic methods. The ind ividual subunits of the enzyme can also be expressed in the Sf9 cells, but the ability to reconstitute active enzyme from extracts containin g individual subunits is quite low. In contrast, the enzyme produced b y coexpression of the two subunits is fully active and retains the pro perties of the mammalian form, including the specificity for the COOH- terminal amino acid of substrate proteins and the ability to bind shor t peptides encompassing the prenylation site of a ras protein. Further more, through atomic absorption analysis of the purified protein, we h ave confirmed the previous tentative assignment of protein farnesyltra nsferase as a zinc metalloenzyme by demonstrating that it contains an essentially stoichiometric amount of zinc. The ability to produce and purify milligram quantities of protein farnesyltransferase readily wil l allow detailed mechanistic and structural studies on this enzyme.