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 Cassidy, Wu Hong-yu, C. Dale Poulter

Research output: Contribution to journalArticle

47 Citations (Scopus)

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

Fingerprint

Saccharomyces cerevisiae
Escherichia coli
Mating Factor
Plasmids
Protein Prenylation
Genes
Proteins
Fungal Proteins
Initiator Codon
Terminator Codon
Ion Exchange Chromatography
Enzymes
Tubulin
Operon
Ribosomes
Recombinant Proteins
Codon
Chromatography
Epitopes
Yeasts

Keywords

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

ASJC Scopus subject areas

  • Genetics

Cite this

Protein farnesyltransferase : production in Escherichia coli and imrmmoaffinity purification of the heterodimer from Saccharomyces cerevisiae. / Mayer, Matthias P.; Prestwich, Glenn D.; Dolence, Julia M.; Cassidy, Pamela; Hong-yu, Wu; Dale Poulter, C.

In: Gene, Vol. 132, No. 1, 30.09.1993, p. 41-47.

Research output: Contribution to journalArticle

Mayer, Matthias P. ; Prestwich, Glenn D. ; Dolence, Julia M. ; Cassidy, Pamela ; Hong-yu, Wu ; Dale Poulter, C. / Protein farnesyltransferase : production in Escherichia coli and imrmmoaffinity purification of the heterodimer from Saccharomyces cerevisiae. In: Gene. 1993 ; Vol. 132, No. 1. pp. 41-47.
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