Abstract
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.
Highlights
The Saccharomycescerevisiae mating pheromone membranes would be greatly facilitated by the ability to purify precursor, prepro-a-factor, can be translocated across chemical amounts of those substrates which can be transyeast endoplasmic reticulum membranes post-translationally inan in vitro system
Efforts have been limited by the inability to isolate sufficient quantities of such secretory protein precursors in a translocation-competent form
The construct was expressed at high levels in a SecYstrain of Escherichia coli
Summary
The Saccharomycescerevisiae mating pheromone membranes would be greatly facilitated by the ability to purify precursor, prepro-a-factor, can be translocated across chemical amounts of those substrates which can be transyeast endoplasmic reticulum membranes post-translationally inan in vitro system. This characteristic makes prepro-a-factor potentially useful as a probe in the biochemical dissection of the mechanismof this basic cellular process. The biochemical characterization of the elements involved that can stably bind to the affinity column under even the in the translocationof proteins across endoplasmic reticulum harshest of denaturing conditions. B containing 0.5 M imidazole, pH 8.0, and with buffer F (6 M guanidine-HC1, 0.2 M acetic acid) to detach any proteins remaining on the column
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