title: Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: application to Bruton's tyrosine kinase.
abstract: Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin-binding peptide (CBP), and it allows for recovery of 20-30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP-His₆ purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems.
authors.affiliation: Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Li
authors.forename: Sarah
authors.initials: S
authors.lastname: Franklin
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Zhang
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Vondriska
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D002352:Carrier Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity; D057809:HEK293 Cells; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D046912:Multiprotein Complexes / Q000302:isolation & purification; D009842:Oligopeptides / Q000737:chemistry*; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
chemical_list: D002352:Carrier Proteins; C471213:His-His-His-His-His-His; D046912:Multiprotein Complexes; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C452700:streptavidin-binding peptide; D006639:Histidine; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human