[17] Expression, purification, and biochemical properties of rabkinesin-6 domains and their interactions with Rab6A

This chapter describes the purification of several Rabkinesin-6 domains from bacteria and eukaryotic systems, and their biochemical characterization (ATPase, MT-binding, hydrodynamic properties).The chapter describes an assay to visualize the interaction between Rab6A and Rabkinesin-6. For purification, Escherichia coli strain freshly transformed with pET-Nt or pTrc-Q665 plasmids are cultured. Bacterial pellets are obtained are sonicated, centrifuged, and chromatographed. Protein fractions are analyzed by SDS-PAGE and Western blot. This protocol leads to the purification of about 0.5–1 mg fusion proteins. α/β-Tubulin is purified from bovine brain with two cycles of polymerization in the presence of GTP, and microtubule (MT)-associated proteins (MAPs) are removed by phosphocellulose chromatography. Tubulin concentration is estimated by the Bradford assay and MT concentration is expressed per tubulin heterodimer. MT-induced ATPase activity of kinesins is often measured by using a NADH coupled enzymatic assay.