Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes.
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.
Zeghouf
Mahel
M
Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, Canada M5G 1L6.
Li
Joyce
J
Butland
Gareth
G
Borkowska
Anna
A
Canadien
Veronica
V
Richards
Dawn
D
Beattie
Bryan
B
Emili
Andrew
A
Greenblatt
Jack F
JF
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*
D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D019473:Transcription Factors, TFII; C056604:transcription factor TFIIF