Characterization of yeast geranylgeranyl transferase type I expressed in Escherichia coli

Hyunkyung Kim, Seung-Hoi Koo, Hee Jung Choi, Chul Hak Yang

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Protein geranylgeranyl transferase type I (GGTase-I) transfers the geranylgeranyl group from geranylgeranyl pyrophosphate (GGPP) to the, cysteine residue of the carboxy-terminal sequence, -Cys-A-A-Leu (A is an aliphatic amino acid) of cytoplasmic proteins. The ram2 and call genes which encode the α and β subunits of yeast GGTase-I respectively were obtained by a polymerase chain reaction (PCR). The ram2 gene was cloned to the pFC vector, which has an chloroamphenicol resistance gene, and the call gene was cloned to the pFlag vector, which has a ampicillin resistance gene. These two recombinant plasmids were co-transformed and expressed in E. coli. GGTase-I was partially purified by ammonium sulfate fractionation followed by Q-Sepharose anion exchange chromatography. The enzyme was identified through immunoblotting assay and characterization studies were performed using active fractions from Q-Sepharose. The partially purified yeast GGTase-I expressed in E. coli showed a dose-dependent increase in the transferase activity, and its optimum pH ranges were found between 6.9 and 7.3. Apparent Km value for GST-PEP of GGTase-I was 1.43 μM and apparent Km value for GGPP of GGTase-I was 0.01 μM. GGTase-I required Mg2+ and Zn2+ for its activity and Zn2+ seemed to be tightly bound to the enzyme.

Original languageEnglish
Pages (from-to)602-608
Number of pages7
JournalMolecules and Cells
Volume6
Issue number5
Publication statusPublished - 1996 Oct 31
Externally publishedYes

Fingerprint

Transferases
Yeasts
Escherichia coli
Genes
Sepharose
Ampicillin Resistance
Ammonium Sulfate
Enzymes
geranylgeranyltransferase type-I
Immunoblotting
Anions
Cysteine
Chromatography
Proteins
Plasmids
Fatty Acids
Amino Acids
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

Characterization of yeast geranylgeranyl transferase type I expressed in Escherichia coli. / Kim, Hyunkyung; Koo, Seung-Hoi; Choi, Hee Jung; Yang, Chul Hak.

In: Molecules and Cells, Vol. 6, No. 5, 31.10.1996, p. 602-608.

Research output: Contribution to journalArticle

Kim, Hyunkyung ; Koo, Seung-Hoi ; Choi, Hee Jung ; Yang, Chul Hak. / Characterization of yeast geranylgeranyl transferase type I expressed in Escherichia coli. In: Molecules and Cells. 1996 ; Vol. 6, No. 5. pp. 602-608.
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