Purification and characterization of recombinant human translation initiation factor eIF3.

Abstract

Eukaryotic translation initiation factor 3 (eIF3) is an essential factor in protein synthesis. In mammals, it is a ~800 kDa complex composed of 12-13 subunits. Biochemical and mechanistic studies of the function of eIF3 and its individual subunits in translation require purified eIF3. However, current strategies for obtaining mammalian eIF3 rely on purification of the endogenous factor from cultured human cells or rabbit reticulocytes lysates, both of which are expensive and time consuming. Here we present a recombinant insect-cell expression and purification system for human eIF3 which allows the purification of large amounts of functional, homogeneous eIF3 efficiently and cost-effective, while also enabling engineering, such as generation of site-specific mutations in the factor for functional studies.