From the Sér
aphin lab at the European
Molecular Biology Laboratory (EMBL).
Content
Introduction
We have developed a new affinity tag and a purification strategy that allow efficient and reliable recovery of proteins present at low cellular level under native conditions: the TAP method.
The procedure is relatively rapid and cheap.
Because it is generic and rapid, the TAP procedure constitutes an important new tool for protein complex characterization and proteome exploration.
As an example, you can see on the right the result of the
purification of the yeast U1 snRNP, a multi-subunit RNA-protein complex
involved in pre-mRNA splicing. Even though this complex is not very
abundant (estimated at 500 copies per cells (Riedel et al. (1986) Proc. Natl. Acad. Sci. U.S.A.
83, 8097-8101), with the efficient TAP method
only 2 liters of yeast culture were required to detect all the yeast U1
snRNP specific proteins by Coomassie staining (arrows).
See Rigaut et al. (1999) Nature Biotech. 17, 1030 -
1032 for details.
TAP strategy overview
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The TAP purification method involves the fusion of the T AP tag to the target protein of interest and the introduction of the construct into the cognate host cell or organism. To purify protein complexes it is preferable to maintain expression of the fusion protein at, or close to, its natural level. The fusion protein present in extracts prepared from these cells, as well as associated components, are then recovered by Tandem Affinity Purification (TAP).
Combined with the highly sensitive mass-spectrometry methods available nowadays, the TAP method is useful to characterize protein complexes. In addition, the activity of the purified proteins can be analyzed.
Tag structure
A preliminary screening indicated that IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the Calmodulin Binding Peptide (CBP) were the best among several tags tested for purification of low abundance proteins. These tags have sufficient affinity for quantitative recovery of fusion protein present at a low level in a complex mixture.
These two tags were fused in tandem separated by a TEV protease cleavage site allowing for Tandem Affinity Purification (TAP).
The TAP tag can be split in two halves that may not be fused to the same
protein: CBP (TAP-C) can be fused to one protein while a TEV protease
cleavage site and ProtA (TAP-A) can be fused to a second protein. This
strategy may be useful, e.g., when some proteins belong to several
complexes or when complex formation is transient. (see Caspary et al. (1999) EMBO J., 18,
3463-3474.)
Construction of recombinant cells or organisms expressing the TAP-tagged
target protein
The TAP purification method involves the fusion of the TAP tag to the target protein of interest and the introduction of the construct to the cognate host cell or organism. If the protein belongs to a complex, it is generally preferable to maintain expression of the fusion protein at, or close to, its natural level. Various strategies are available depending of the host cell or organism. Obviously, if the goal is to purify associated proteins, the target protein should be expressed in cells or organisms expressing naturally the complex to ensure that the other subunits are also expressed.
Preparation of extracts
Preparation of extract is a critical step in the purification process, since this step influences the total quantity of the desired protein recovered and the biological activity of the protein. A number of variables determine the success of a protein extraction procedure: cell lysis method, presence of protease inhibitors, choice of buffers, etc…
Tandem Affinity Purification (TAP)
Optimal conditions for every step of the purification have been determined in test experiments. These optimized conditions were combined to generate the final optimized TAP protocol.
The fusion protein and associated components are recovered from extracts by affinity selection on an IgG-matrix. After washing, the TEV protease is added to release the bound material. The eluate is incubated with calmodulin-coated beads in the presence of calcium. This second affinity step is required to remove the TEV protease as well as traces of contaminants remaining after the first affinity selection. After washing, the bound material is released with EGTA. The TAP protocol is available here.
Protein analysis by SDS-PAGE
SDS-(Sodium dodecyl sulfate) polyacrylamide gel electrophoresis (SDS-PAGE) is used for separating the subunits of purified protein complexes.
SDS-PAGE requires concentrated and relatively salt-free protein samples.
Protein identification
Proteins can be efficiently identified by mass spectrometry. Several methods have been developed in the EMB L Protein & Peptide Group over the last few years. If sufficient amounts of proteins are available, Edman degradation would be another alternative. One can also probe a blot of the purified proteins with antibodies.
Functional assays
Because proteins are purified under native conditions with the TAP method, the purified material can also be used for biochemical analysis and functional assays. This is particularly useful to demonstrate that the function of a putative protein was correctly predicted.
Alternative assays
Because complexes are purified under native conditions with the TAP method, the presence of different kinds of molecules associated with the TAP-tagged target protein (RNA, metabolites, lipids, etc…) can also be tested.
Primary reference
Publications reporting results obtained with the TAP strategy should refer to:
A generic protein purification method for protein complex characterization and proteome exploration
Some publications reporting usage of the TAP method
Author: seraphin@cgm.cnrs-gif.fr Last Modified, February 27, 2002
