Sequential peptide affinity purification system for the systematic isolation and identification of protein complexes from Escherichia coli.
Biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. Accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. To this end, a sequential peptide affinity (SPA) purification system was developed to enable the rapid and efficient isolation of native Escherichia coli protein complexes (J Proteome Res 3:463-468, 2004). SPA purification makes use of a dual-affinity tag, consisting of three modified FLAG sequences (3X FLAG) and a calmodulin binding peptide (CBP), spaced by a cleavage site for tobacco etch virus (TEV) protease (J Proteome Res 3:463-468, 2004). Using the lambda-phage Red homologous recombination system (PNAS 97:5978-5983, 2000), a DNA cassette, encoding the SPA-tag and a selectable marker flanked by gene-specific targeting sequences, is introduced into a selected locus in the E. coli chromosome so as to create a C-terminal fusion with the protein of interest. This procedure aims for near-endogenous levels of tagged protein production in the recombinant bacteria to avoid spurious, non-specific protein associations (J Proteome Res 3:463-468, 2004). In this chapter, we describe a detailed, optimized protocol for the tagging, purification, and subsequent mass spectrometry-based identification of the subunits of even low-abundance bacterial protein complexes isolated as part of an ongoing large-scale proteomic study in E. coli (Nature 433:531-537, 2005).
Babu
Mohan
M
Banting and Best Department of Medical Research, University of Toronto, Donnelly Center for Cellular and Biomolecular Research, 160 College Street, Toronto, ON, Canada M5S 3E1.
Butland
Gareth
G
Pogoutse
Oxana
O
Li
Joyce
J
Greenblatt
Jack F
JF
Emili
Andrew
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010449:Peptide Mapping; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods*
D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; C099488:TEV protease