Sequential Peptide Affinity (SPA) System for the Identification of Mammalian and Bacterial Protein Complexes

Abstract

A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system). By avoiding overproduction of the target protein, this system allows for the efficient purification of natural protein complexes and their identification by mass spectrometry. We also present an adaptation of the SPA system for the efficient purification and identification of protein complexes in E. coli and, potentially, other bacteria. Keywords: SPA system • protein complexes • protein−protein interactions • affinity purification • mass spectrometry • human cell • E. coli