A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes

Abstract

Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled an efficient and large-scale purification of native protein complexes. However, the TAP tag features a size of 21 kDa and requires time consuming cleavage. By combining a tandem Strep-tag II with a FLAG-tag we were able to reduce the size of the TAP (SF-TAP) tag to 4.6 kDa. Both moieties have a medium affinity and avidity to their immobilised binding partners. This allows the elution of SF-tagged proteins under native conditions using desthiobiotin in the first step and the FLAG octapeptide in the second step. The SF-TAP protocol represents an efficient, fast and straightforward purification of protein complexes from mammalian cells within 2.5 h. The power of this novel method is demonstrated by the purification of Raf associated protein complexes from HEK293 cells and subsequent analysis of their protein interaction network by dissection of interaction patterns from the Raf binding partners MEK1 and 14-3-3.

Elution Profile Analysis of SDS-induced Subcomplexes by Quantitative Mass Spectrometry

“…The more stable binding of dynein light chain, as well as of the 14 -3-3 protein isoforms, compared with IFT proteins, is in-line with the fact that these were among the few proteins that could previously be identified by lebercilin SF-TAP purification (9).…”

“…Previous studies have shown that the 14 -3-3 proteins can form a stable complex (9,22). Relative to the other subcomplexes, here the 14 -3-3 proteins form a less homogeneous group of elution profiles (EPD 14 -3-3 Յ0.071, n ϭ 4).…”

“…The reference proteins for possible submodules within the 14-3-3-complex (supplemental Table S2) were selected from the literature (9,25) and the String interaction database (www.string-db.org). The destabilization of the protein complex of 14-3-3-showed several submodules eluting from the complex (Fig.…”

Modification, Development, Application and Prospects of Tandem Affinity Purification Method

“…The combination of a streptavidin-binding peptide (SBP) and a CBP has been verified in human cells (Ahmed et al, 2010;Colpitts et al, 2011). Recently, use of another alternative tandem affinity tag, composed of two Strep-tag II and a FLAG-tag (SF), has been published (Gloeckner et al, 2007). This SF tag reduced the size of the TAP tag to 4.6 kDa.…”

“…The CBP affinity step has been proved to be problematic in that case where many endogenous proteins of mammalian cells interact with calmodulin in a calcium-dependent manner (Agell et al, 2002;Head, 1992). A simple alternative solution is replacing the CBP tag with other affinity tags, such as the FLAG sequence (Gloeckner et al, 2007;Knuesel et al, 2003), ProtC and biotinylation tag (Drakas et al, 2005). The main challenge of the TAP strategy comes from the competition of endogenous proteins with the tagged protein in protein complex assembly.…”