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.
Mayer
M P
MP
Department of Chemistry, University of Utah, Salt Lake City 84112.
Prestwich
G D
GD
Dolence
J M
JM
Bond
P D
PD
Wu
H Y
HY
Poulter
C D
CD
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005800:Genes, Fungal; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014166:Transferases / Q000235:genetics* / Q000302:isolation & purification; D014404:Tubulin / Q000235:genetics
D004277:DNA, Single-Stranded; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)