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.

Figure 1

Schematic representation of the affinity-tagged human Btk. Btk was N-terminally fused to the CBP-SBP tandem tag system and a C-terminal His-tag engineered to expand protein purification options. The positively and negatively charged residues in CBP and SBP, respectively, are highlighted. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Western blot analysis of human Btk expression and extraction. Whole cell lysate (WCL) of pNTAP-hBtk- or empty vector- (pNTAP-null) transfected HEK-293 cells were separated along with supernatant (sup) or pellet (pel) extraction from pNTAP-hBtk transfected cells, followed by immunoblotting for Btk. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3

Dual streptavidin- and calmodulin-based purification. A: First step streptavidin-based purification recovers substantial Btk protein: supernatant of whole cell lysate (WCL); unbound portion (SBP-Unb.); and product of 2 mM biotin eluate (Bio-Elu.). B: Protein recovery as illustrated by serial dilution of biotin eluate: undiluted (Bio-Elu.); twofold, fourfold, sixfold, and eightfold dilution; and supernatant of whole cell lysate (WCL). C: Second phase of TAP purification via calmodulin binding is ineffective to recover Btk: biotin eluate resulted from the first round of purification (Bio-Elu.); unbound portion following incubation of streptavidin eluate with calmodulin resin (CBP-Unb.); product of 5 mM EGTA elution (EGTA-Elu.). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4

Dual streptavidin- and nickel-based purification. A: Eluate from first step streptavidin-based purification (Bio-Elu.); unbound portion (His-Unb.); and product of 300 mM imidazole eluate (Imi-Elu.). B: Protein recovery, including Biotin eluate (Bio-Elu.) and four-fold diluted imidazole eluate (Imi-Elu. 4). C: Silver staining of partially and fully purified Btk complex: biotin eluate from the first round of purification (Bio-Elu.) and imidazole eluate from the second round of purification (Imi-Elu.). Asterisk indicates Btk as determined by mass spectrometry. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 5

Methodology for analysis of Btk-associated proteins using new TAP vector combined with mass spectrometry. A: Oriole staining of tandem affinity purified Btk complex: negative control of tandem affinity purification (TAP-Null) and tandem affinity purified Btk complex (TAP-Btk). Asterisk indicates Btk. B: Experimental workflow for TAP purification and protein identification.