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

Yifeng Li, Sarah Franklin, Michael J. Zhang, Thomas M. Vondriska

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

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 calmodulinbinding 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.

Original languageEnglish (US)
Pages (from-to)140-149
Number of pages10
JournalProtein Science
Volume20
Issue number1
DOIs
StatePublished - Jan 2011

Keywords

  • Btk
  • Calmodulin-binding peptide
  • His-tag
  • Protein complex
  • Streptavidinbinding peptide
  • Tandem affinity purification

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