Abstract
This chapter describes a method to geranylgeranylate recombinant Rab proteins directly in E. coli lysates, without the need to purify REP or GGTase II. The procedure can be used on an analytical scale to rapidly determine whether amino acid substitutions affect Rab prenylation. Alternatively, it can be scaled up to prepare modified recombinant proteins in microgram quantities sufficient for functional studies in reconstituted systems. Cyclic DNA encoding the Rab protein of interest is cloned into a prokaryotic expression vector for protein production in E. coli BL21(DE3)pLysS. To facilitate purification and immunodetection of the substrate proteins, the rab cDNA sequence can be modified by polymerase chain reaction (PCR) to add an epitope tag, such as His 6 (hexahistidine) or myc (EQKLISEEDL) to the amino terminus of the protein. Such modifications do not appear to affect the efficiency of Rab prenylation or subcellular localization. Expression of the recombinant Rab protein is induced by exposing the E. coli to 1.0 mM isopropyl- β -D-thiogalactopyranoside (IPTG) for 1–2 hr, followed by centrifugation of the cells. The procedures for expression of recombinant Rab proteins in E. coli have been described in the chapter.
Published Version
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