Characterization of yeast geranylgeranyl transferase type I expressed in Escherichia coli

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.