Tandem Affinity Purification
The tandem affinity purification (TAP ) discloses protein purification using two different chromatographic purification methods. When using two different protein tags, for example, two different affinity chromatography to be performed. In a broader sense, the Tandem Affinity Purification includes all serial purification methods based on the affinity of the material to be purified stationary phase.
TAP tag
Towards the end of the second millennium Tandem Affinity Purification a day was developed for proteins, which consists of two different consecutive protein tags. Its outer (N- or C -terminal ) protein tag is cleaved in the first column after removal of undesirable constituents from the cell digestion by the protease TEV, whereby the desired protein ( without its outer protein tag ), the TEV protease and some residual contaminants from the chromatography column to elute. The TEV - protease from the tobacco etch virus has a longer recognition sequence (Glu -Asn -Leu- Tyr -Phe -Gln- (Gly / Ser ) or ENLYFQ (G / S) ) to be purified, the protein not at other locations cut. Usually is used, the CAM -based tag or a biotin tag, since the non-denaturing elution conditions are, and the components of the elution buffer (such as EGTA, biotin) for removal of the TEV protease, and residual contaminants as a second, inboard protein tag a little disturbing in a subsequent use.
The choice of the TAP tag and the N- or C-terminal insertion is determined by the conservation of biological activity of the protein, since some combinations in a defective protein folding and thus impaired function may result. Therefore, the influence of the position of the TAP - tag is determined in the function of the fusion protein experiments.
After only a few combinations allow high amounts of protein and yields of protein purification, today scattered combinations are used, for example, the SBP -CBP tag ( Interplay ), the FLAG -HA tag, the 3xFLAG - His-tag, the ProtA - ProtC tag that protg SBP - tag, which 2xFLAG ProtA - tag, the His - tag 2xStrepII, the SBP -HA tag and the SBP- His tag.
Applications
The tandem affinity purification is used, inter alia, to reduce the cleaning steps in the protein purification. In combination with mass spectrometry or Western blotting can identify proteins and protein -protein interactions longer dwell time will be detected.