Purification of Prefoldin

Abstract

Prefoldin is a recently discovered chaperone protein (1, 2) that functions by directing unfolded target proteins to the cytosolic chaperonin, c-cpn (3 4). It thus serves to promote productive folding in the crowded molecular environment that prevails inside living cells, where there are many competing pathways for nonnative proteins. Prefoldin binds to c-cpn and transfers its target protein to the chaperonin (1). Like c-cpn, prefoldin is heteromeric: it is assembled from six different polypeptides in the size range 14-23 kDa. Each subunit is present in approximately stoichiometric amount. Prefoldin migrates with an apparent molecular mass of about 200 kDa on gel filtration, so each molecule contains either one or two each of the six subunit polypeptides. The prospect of engineering the simultaneous expression of six open reading frames in a prokaryotic host is a daunting one; in any event, it is uncertain whether the expressed subunits would assemble into functional prefoldin molecules in Escherichia coli. From a practical point of view, therefore, a biochemical study of prefoldin function requires its purification from a tissue source. Prefoldin is widely expressed; however, the protocol described here is for purification from bovine testis, because (1) testis tissue expresses relatively high levels of prefoldin and c-cpn, and (2) the tissue is easily obtainable from slaughterhouses and is relatively inexpensive. The purification procedure involves the preparation of a soluble protein extract, followed by ammonium sulfate fractionation and (minimally) four successive chromatographic dimensions.