Expression of mammalian geranylgeranyltransferase type-II in Escherichia coli and its application for in vitro prenylation of Rab proteins

摘要

Mammalian geranylgeranyltransferase type II (GGTase-II) is a 100-kDa heterodimer that catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab GTPases. This modification is essential for the biological activity of Rab proteins. Geranylgeranylation can be performed in vitro using recombinant GGTase-II but so far large-scale production of the enzyme was challenging. We report here the design of a two plasmid expression system that will produce GGTase-II at levels as high as 15 mg/L in Escherichia coil, The protein was produced as a heterodimer with the cu subunit bearing a cleavable tandem 6His-glutathione S-transferase (G:ST) tag that was used for two-step purification of the enzyme. Purified enzyme was functionally active as determined by in vitro prenylation and phosphoisoprenoid binding assay. Furthermore, the c;ST-tagged GGTase-II was used for preparative in vitro prenylation of the Rab7:REP-1 complex. Using this procedure, 10 mg of doubly prenylated Rab7:REP-1 complex were obtained. (C) 2001 Academic Press. [References: 16]