1] Purification of His6-tagged Rab1 proteins using bacterial and insect cell expression systems

Abstract

Publisher Summary This chapter describes the isolation of recombinant wild-type or mutant forms of Rab1 via expression in Escherichia coli ( E.coli ) and Spodoptera frugiperda (Sf9) insect cells. Although the bacterial expression system is more convenient from a technical point of view, the utility of Rab1 proteins prepared from E. coli is limited by the fact that these invariably lack the COOH-terminal geranylgeranyl (GG) groups that are essential for normal Rab1 function. In contrast, the eukaryotic expression system allows the purification of membrane-associated, isoprenylated forms of the proteins (Rab1GG). Both expression systems require the purification of relatively minor pools of functional protein. In the case of E. coli , this is because of the strong tendency of Rab1 proteins to form inclusion bodies. To obtain the active forms of the proteins, the chapter focuses on the purification of the soluble pool that represents no more than 1–10% of the total production. In Sf9 cells, the yields of isoprenylated Rab1 proteins are low as