Preparation of Recombinant Rab Geranylgeranyltransferase and Rab Escort Proteins

This chapter describes techniques for the expression and purification of Rab geranylgeranyltransferase (GGTase) and Rab escort protein (REP) in insect Spodoptera frugiperda (Sf9) cells using a baculovirus expression system that allows the production of milligram quantities of active components for the prenylation of Rab proteins. The purified components can be used in vitro to prenylate all Rab proteins tested so far, regardless of the cysteine motif present at the COOH terminus. Small GTPases of the Ras superfamily—including Rab proteins—are modified at their COOH termini by the covalent attachment of a prenyl group to a cysteine residue. Two types of prenyl groups—C_l5 farnesyl and C₂₀ geranylgeranyl (GG)—have been found attached to proteins. Genetic experiments in yeast have demonstrated the physiological importance of Rab proteins in intracellular vesicular transport, but their precise biochemical role remains elusive. A number of laboratories have developed in vitro systems that reproduce intracellular vesicular movements that normally occur during exocytosis and endocytosis. The chapter describes methods for the production of recombinant Rab GGTase and REPs and their use to modify Rab proteins in vitro.