PROTEIN FARNESYLTRANSFERASE - PRODUCTION IN ESCHERICHIA-COLI AND IMMUNOAFFINITY PURIFICATION OF THE HETERODIMER FROM SACCHAROMYCES-CEREVISIAE

Protein farnesylation in Saccharomyces cerevisiae is mediated by a het erodimeric enzyme, protein farnesyltransferase (PFTase), encoded by th e genes RAM1 and RAM2. A series of plasmids for the expression of RAM1 and RAM2 in Escherichia coli was prepared and evaluated. Maximal prod uction of functional PFTase was seen in strains containing a multicopy plasmid with a synthetic operon in which the RAM1 and RAM2 structural genes were translationally coupled by overlapping TAATG stop-start co dons and by locating a ribosome-binding site near the 3' end of the up stream gene. This was accomplished by an insertional mutation at the 3 '-end of RAM1 that embedded an AGGAGGAG sequence within codons for the tetrapeptide, QEEF, added to the end of the Ram1 protein. The QEEF C- terminal motif in the Raml subunit of PFTase facilitated purification of the enzyme by immunoaffinity chromatography on an anti-alpha-tubuli n column prepared using monoclonal antibodies that recognized a tripep tide EEF epitope. Heterodimeric recombinant yeast PFTase=QEEF (re-PFTa se=QEEF) constituted approximately 4% of total soluble protein in indu ced cells and was readily purified 25-fold in two steps by ion exchang e and immunoaffinity chromatography in an overall 25% yield. Michaelis constants for farnesyl diphosphate (FPP) and H(ras) protein (modified to contain a yeast a-mating factor PACVIA sequence at the C terminus) were 5.5 and 15 muM, respectively; the k(cat) was 0.7 s-1.