Tandem Affinity Purification

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Scheme of the Tandem Affinity Purification

The tandem affinity purification (TAP) describes the protein purification using two different chromatographic purification methods. If two different protein tags are used , for example two different affinity chromatographies can be carried out. In a broader sense, Tandem Affinity Purification includes all serial purification processes that are based on the affinity of the material to be purified to the stationary phase .

TAP day

From 1999 onwards, Tandem Affinity Purification Tags were developed for proteins, which consist of two different, consecutive protein tags. Its outer ( N- or C- terminal ) protein tag is cleaved in the first column by the protease TEV after removing unwanted components from the cell disruption , whereby the desired protein (without its outer protein tag), the TEV protease and some Elute residual contamination from the chromatography column . 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)) so that the protein to be purified is not transferred to other locations cut. In most cases, the CaM tag or a biotin- based tag is used as the second, internal protein tag to remove the TEV protease and residual contamination , as their elution conditions are non- denaturing and the components of the elution buffer ( EGTA or biotin) are less common in one interfere with the following use.

The choice of the TAP tag and the N- or C-terminal insertion is determined by the preservation of the biological activity of the protein, since some combinations can lead to incorrect protein folding and thus reduced function. Therefore, the influence of the position of the TAP tag on the function of the fusion protein is determined experimentally.

Since only a few combinations allow high amounts of protein and protein purification yields , individual combinations are used today, e.g. B. the SBP -CBP-Tag ( InterPlay ), the FLAG -HA-Tag, the 3xFLAG- His-Tag , the ProtA-ProtC-Tag, the ProtG-SBP-Tag, the 2xFLAG-ProtA-Tag, the His- 2x StrepII-Tag , the SBP-HA-Tag and the SBP-His-Tag.

Applications

The Tandem Affinity Purification is used, among other things, to reduce the purification steps involved in protein purification. In combination with mass spectrometry or with a Western blot , proteins can be identified and protein-protein interactions of longer residence times can be detected.

literature

  • Hubert Rehm , Thomas Letzel: The Experimenter: Protein Biochemistry / Proteomics . 6th edition, Spektrum Akademischer Verlag, Heidelberg 2009. ISBN 978-3-8274-2312-2 .

Individual evidence

  1. ↑ JT Kadonaga, R. Tjian: Affinity purification of sequence-specific DNA binding proteins. In: Proc Natl Acad Sci USA (1986), vol. 83 (16), pp. 5889-5893. PMID 3461465 ; PMC 386402 (free full text).
  2. ↑ M. de Frutos, FE Regnier: Tandem chromatographic-immunological analyzes. In: Anal Chem. (1993), Vol. 65 (1), pp. 17A-25A. PMID 8420386 .
  3. ↑ G. Rigaut, et al .: A generic protein purification method for protein complex characterization and proteome exploration . In: Nature Biotechnology . 17, No. 10, 1999, pp. 1030-1032. doi : 10.1038 / 13732 . PMID 10504710 .
  4. ↑ O. Puig, et al .: The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification . In: Methods . 24, No. 3, 2001, pp. 218-229. doi : 10.1006 / meth.2001.1183 . PMID 11403571 .
  5. ↑ O. Puig, F. Caspary, G. Rigaut, B. Rutz, E. Bouveret, E. Bragado-Nilsson, M. Wilm, B. Séraphin: The tandem affinity purification (TAP) method: a general procedure of protein complex purification. In: Methods (2001), Volume 24 (3), pp. 218-229, PMID 11403571 .
  6. ^ AC Gavin, P. Aloy, P. Grandi, R. Krause et al .: Proteome survey reveals modularity of the yeast cell machinery. In: Nature (2006), Volume 440, pp. 631-636, PMID 16429126 .
  7. ^ A b Y. Li: The tandem affinity purification technology: an overview. In: Biotechnol Lett. (2011), Volume 33 (8), pp. 1487-1499, PMID 21424840 .