title: Preparation of multiprotein complexes from Arabidopsis chloroplasts using tandem affinity purification.
abstract: Since its first description in 1998 (Rigaut et al., Nat Biotech 17:1030-1032, 1999), the TAP method, for Tandem Affinity Purification, has become one of the most popular methods for the purification of in vivo protein complexes and the identification of their composition by subsequent mass spectrometry analysis. The TAP method is based on the use of a tripartite tag fused to a target protein expressed in the organism of interest. A TAP tag has two independent binding regions separated by a protease cleavage site, and therefore allows two successive affinity purification steps. The most common TAP tag consists of two IgG binding repeats of Protein A from Staphylococcus aureus (ProtA) separated from a calmodulin-binding peptide by a Tobacco Etch Virus (TEV) protease cleavage site. Using the TAP method, native protein complexes can be purified efficiently with a reduced contaminant background when compared to single step purification methods. Initially developed in the yeast model system, the TAP method has been adapted to most common model organisms. The first report of the purification of protein complexes from plant tissue by the TAP method was published in 2004 by Rohila et al. (Plant J 38:172-181, 2004). The synthetic TAP tag gene described in this study has been optimized for use in plants, and since then, has been successfully used from single gene analyses to high-throughput studies of whole protein families (Rohila et al., PLoS ONE 4:e6685, 2009). Here, we describe a TAP tag purification method for the purification of protein complexes from total Arabidopsis extracts, that we employed successfully using a TAP-tagged chloroplast outer envelope protein.
authors.affiliation: Plant Physiology Laboratory, University of Neuchatel, Neuchatel, Switzerland.
authors.forename: Charles
authors.initials: C
authors.lastname: Andrès
authors.forename: Birgit
authors.initials: B
authors.lastname: Agne
authors.forename: Felix
authors.initials: F
authors.lastname: Kessler
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000166:cytology* / Q000254:growth & development; D011232:Chemical Precipitation; D002725:Chloroform / Q000737:chemistry; D060365:Chloroplast Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D046508:Culture Techniques; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D008863:Microspheres; D009942:Organometallic Compounds / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
chemical_list: D060365:Chloroplast Proteins; D007074:Immunoglobulin G; D009942:Organometallic Compounds; C515601:Sypro Ruby; D002725:Chloroform; D012685:Sepharose; D010450:Endopeptidases; C099488:TEV protease; D014357:Trypsin; D000432:Methanol