title: Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes.
abstract: A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system). By avoiding overproduction of the target protein, this system allows for the efficient purification of natural protein complexes and their identification by mass spectrometry. We also present an adaptation of the SPA system for the efficient purification and identification of protein complexes in E. coli and, potentially, other bacteria.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, Canada M5G 1L6.
authors.forename: Mahel
authors.initials: M
authors.lastname: Zeghouf
authors.forename: Joyce
authors.initials: J
authors.lastname: Li
authors.forename: Gareth
authors.initials: G
authors.lastname: Butland
authors.forename: Anna
authors.initials: A
authors.lastname: Borkowska
authors.forename: Veronica
authors.initials: V
authors.lastname: Canadien
authors.forename: Dawn
authors.initials: D
authors.lastname: Richards
authors.forename: Bryan
authors.initials: B
authors.lastname: Beattie
authors.forename: Andrew
authors.initials: A
authors.lastname: Emili
authors.forename: Jack F
authors.initials: JF
authors.lastname: Greenblatt
mesh_terms: D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006801:Humans; D013058:Mass Spectrometry; D046912:Multiprotein Complexes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D019473:Transcription Factors, TFII; C056604:transcription factor TFIIF