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J Biol Chem

Title:		Secretion in yeast. Purification and in vitro translocation of chemical amounts of prepro-alpha-factor
Author(s):		Bush GL; Tassin AM; Friden H; Meyer DI;
Address:		"Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024"
Journal Title:		J Biol Chem
Year:		1991
Volume:		266
Issue:		21
Page Number:		13811 - 13814
DOI:
ISSN/ISBN:		0021-9258 (Print) 0021-9258 (Linking)
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"
Keywords:		"Base Sequence Biological Transport Cell-Free System Chromatography, Affinity DNA Mutational Analysis Escherichia coli/genetics Mating Factor Microsomes/metabolism Molecular Sequence Data Nickel/chemistry Oligonucleotides/chemistry Peptides/genetics/*metab;"
Notes:		"MedlineBush, G L Tassin, A M Friden, H Meyer, D I eng GM07185/GM/NIGMS NIH HHS/ GM38538/GM/NIGMS NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 1991/07/25 J Biol Chem. 1991 Jul 25; 266(21):13811-4"