The Saccharomyces cerevisiae mating pheromone precursor, prepro-alpha-factor, can be translocated across yeast endoplasmic reticulum membranes post-translationally in an in vitro system. This characteristic makes prepro-alpha-factor potentially useful as a probe in the biochemical dissection of the mechanism of this basic cellular process. Efforts have been limited by the inability to isolate sufficient quantities of such secretory protein precursors in a translocation-competent form. We report here the one-step purification of chemical amounts of translocation-competent prepro-alpha-factor using nickel ion affinity chromatography on nitrilotriacetate resin. An oligonucleotide encoding 6 histidine residues was inserted into a genomic clone encoding prepro-alpha-factor 5' of the naturally occurring translational stop codon by site-directed mutagenesis. The construct was expressed at high levels in a SecY- strain of Escherichia coli. The produced preprotein was solubilized in 6 M guanidine hydrochloride and bound to nitrilotriacetate resin. Prepro-alpha-factor was recovered at a purity in excess of 95% by elution with 0.25 M imidazole, 8 M urea, which competitively displaced the histidine affinity tag from the nickel column. The chemical amounts of prepro-alpha-factor obtained in this way were determined to be competent for translocation across yeast microsomal membranes and for subsequent modifications such as signal sequence cleavage and N-linked glycosylation.

• PMID: 1856213
• PubMed
• PubTator

Reference Type

Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Bush GL, Tassin AM, Fridén H, Meyer DI

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