Protein farnesyltransferase: production in Escherichia coli and imrmmoaffinity purification of the heterodimer from Saccharomyces cerevisiae

Matthias P. Mayer, Glenn D. Prestwich, Julia M. Dolence, Pamela D. Bond, Wu Hong-yu, C. Dale Poulter

Research output: Contribution to journalArticle

47 Scopus citations

Abstract

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-α-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 μM, respectively; the kcat was 0.7 s-1.

Original languageEnglish (US)
Pages (from-to)41-47
Number of pages7
JournalGene
Volume132
Issue number1
DOIs
StatePublished - Sep 30 1993
Externally publishedYes

Keywords

  • Farnesyl diphosphate
  • H protein
  • Michaelis constants
  • prenylation
  • yeast

ASJC Scopus subject areas

  • Genetics

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