High level expression of mammalian protein farnesyltransferase in a baculovirus system. The purified protein contains zinc.
The mammalian enzyme protein farnesyltransferase is a heterodimeric protein that catalyzes the addition of a farnesyl isoprenoid to a cysteine 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 been 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 co-expression of the two subunits of protein farnesyltransferase by Sf9 cells infected with a recombinant baculovirus containing the coding sequences of both polypeptides. This results in the production of milligram quantities of enzyme which can be readily purified by conventional chromatographic methods. The individual subunits of the enzyme can also be expressed in the Sf9 cells, but the ability to reconstitute active enzyme from extracts containing individual subunits is quite low. In contrast, the enzyme produced by co-expression of the two subunits is fully active and retains the properties of the mammalian form, including the specificity for the COOH-terminal amino acid of substrate proteins and the ability to bind short peptides encompassing the prenylation site of a ras protein. Furthermore, through atomic absorption analysis of the purified protein, we have confirmed the previous tentative assignment of protein farnesyltransferase 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 will allow detailed mechanistic and structural studies on this enzyme.
Chen
W J
WJ
Department of Molecular Biology, Glaxo, Inc. Research Institute, Research Triangle Park, North Carolina 27709.
Moomaw
J F
JF
Overton
L
L
Kost
T A
TA
Casey
P J
PJ
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D003035:Cobalt / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009036:Moths; D010957:Plasmids; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014166:Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000032:analysis* / Q000494:pharmacology
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D003035:Cobalt; D004492:Edetic Acid; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D016283:Proto-Oncogene Proteins p21(ras); D015032:Zinc; D002118:Calcium