title: Secretion in yeast. Purification and in vitro translocation of chemical amounts of prepro-alpha-factor.
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.
authors.affiliation: Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024.
authors.forename: G L
authors.initials: GL
authors.lastname: Bush
authors.forename: A M
authors.initials: AM
authors.lastname: Tassin
authors.forename: H
authors.initials: H
authors.lastname: Fridén
authors.forename: D I
authors.initials: DI
authors.lastname: Meyer
mesh_terms: D001483:Base Sequence; D001692:Biological Transport; D002474:Cell-Free System; D002846:Chromatography, Affinity; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D000072235:Mating Factor; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D014158:Transcription, Genetic
chemical_list: D009841:Oligonucleotides; D010455:Peptides; D011994:Recombinant Proteins; D000072235:Mating Factor; D009532:Nickel