Protein farnesyltransferase: production in Escherichia coli and immunoaffinity purification of the heterodimer from Saccharomyces cerevisiae.
Protein farnesylation in Saccharomyces cerevisiae is mediated by a heterodimeric enzyme, protein farnesyltransferase (PFTase), encoded by the genes RAM1 and RAM2. A series of plasmids for the expression of RAM1 and RAM2 in Escherichia coli was prepared and evaluated. Maximal production 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 codons and by locating a ribosome-binding site near the 3' end of the upstream 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 Ram1 subunit of PFTase facilitated purification of the enzyme by immunoaffinity chromatography on an anti-alpha-tubulin column prepared using monoclonal antibodies that recognized a tripeptide EEF epitope. Heterodimeric recombinant yeast PFTase::QEEF (re-PFTase::QEEF) constituted approximately 4% of total soluble protein in induced cells and was readily purified 25-fold in two steps by ion exchange and immunoaffinity chromatography in an overall 25% yield. Michaelis constants for farnesyl diphosphate ( FPP) and Hras protein (modified to contain a yeast a-mating factor PACVIA sequence at the C terminus) were 5.5 and 15 microM, respectively; the kcat was 0.7 s-1.[1]References
- Protein farnesyltransferase: production in Escherichia coli and immunoaffinity purification of the heterodimer from Saccharomyces cerevisiae. Mayer, M.P., Prestwich, G.D., Dolence, J.M., Bond, P.D., Wu, H.Y., Poulter, C.D. Gene (1993) [Pubmed]
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