Expression and purification of recombinant N-ethylmaleimide-sensitive fusion protein from Escherichia coli

This chapter describes techniques for the expression and purification of large quantities of active, recombinant Chinese hamster ovary (CHO) cell NSF from the bacterium Escherichia coll. Availability of unlimited amounts of this protein, and the opportunity to prepare genetically modified derivatives, will greatly facilitate analysis of the biochemical and molecular properties of NSF. The N ethylmaleimide (NEM)-sensitive fusion protein (NSF) is a homotetramer of 76 kDa, initially purified from CHO cells and essential for the fusion of transport vesicles with their cognate acceptor membrane at many stages in the secretory and endocytic pathways. Studies in vitro and in vivo have implicated mammalian NSF and Sec 18p, the Saccharomyces cerevisiae homolog, in endoplasmic reticulum (ER)-to-Golgi traffic, in at least two stages of intra-Golgi transport, and in endocytosis. The chapter discusses the construction of recombinant expression system for NSF. It expresses a derivative of NSF bearing a carboxy-terminal epitope tag because of the lack of anti-NSF antibodies suitable for immunoprecipitation or Western blotting.