Protein Complex Purification and
 Protein:Protein Interaction Analysis


- based on Strep-tag® and One-STrEP-tag technology

"Protein Complex Analysis combining a "one-step co-purification with a mass spectrometry approach"

Rigaut et al., 1999

Procedure


 


 Step 1-3 Protein complex purification; Step 4 Protein complex analyses

1. A tagged bait protein is expressed in the target cell. At a given time point, cells are harvested and lysed.
2. The lysate containing the bait with putatively interacting preys is subjected to tag-based affinity chromatography.
3. Isolated protein complexes are analyzed by SDS-PAGE and silver staining, and compared to mock isolates.
4. Potential preys could be identified by mass spectrometry or verified by Western blots with specific antibodies.

Properties / Benefits

  • Strep-tag®II and One-STrEP-tag have a neutral amino acid composition
  • Purification of recombinant proteins to over 99% purity in a single step
  • Very low tendency of Strep-Tactin® to bind other proteins non-specifically
  • Highly specific Strep-tag®II (OneSTrEP-tag):Strep-Tactin® interaction
  • Specific competitive elution with minute amounts of (desthio-)biotin in the physiological wash buffer


Suitability for PPI
Strep-tag® technology was used from the beginning to purify intact protein complexes in a preparative manner, even if just one subunit carries the tag. For example, the cytochrome C oxidase, an integral membrane protein, was isolated after complexation with a cognate recombinant antibody fragment equipped with the Strep-tag® and the crystal structure of the entire assembly was subsequently determined2.


One-STrEP-tag
The development of a tandem arrangement of two Strep-tag®II sequences, dubbed One-STrEP-tag, even improved performance by increasing purification yields of poorly expressed protein complexes and sustaining elevated detergent concentrations to reduce background.


Strep-tag II: SAWSHPQFEK

One-STrEP-tag: SAWSHPQFEK(GGGS)2GGSAWSHPQFEK

As a conclusion, extremely efficient and fast Strep-tag®II and One-STrEP-tag are reliable tools to meet the challenge of isolating protein complexes at high purity without loosing transient binders.


References:

  1. Schmidt & Skerra, 2007, Nature Protocols 2: 1528-35
  2. Ostermeier et al., 1997, PNAS 94: 10547-10553
  3. Rigaut et al., 1999, Nature Biotech. 17: 1030-103
  4. Juntilla et al., 2005, Proteomics 5: 1199-1203
  5. Johansen et al., 2008, J Cell Sci 121: 854-864
  6. Groth et al. 2007 Science 318: 1928-1931
  7. Schaffitzel & Ban, 2007, J Struct Biol 158: 463-471
  8. Morita et al., 2007, EMBO J. 26: 4215-4227
  9. Morita et al., 2007, Cell Host & Microbe 2: 19-28
  10. Gloeckner et al. 2007 Proteomics 7, 4228-4234
  11. Herzberg et al. 2007 Proteomics 7, 4032-4035
  12. Jarchow et al., 2008, Proteomics, 8: 4987-4994
  13. Glatter and Wepf , 2009, Molecular Systems Biology 5: 237
  14. Pegoraro, 2009, Nature Cell Biology 11 : 1261-1267
  15. Amako et al., 2009 J Virol.; 83(18): 9237–9246
  16. Gianni et al., 2009 J. Biol. Chem. 284: 17370-17382,
  17. Rameix-Welti et al., 2009 J.Virol. 83(3):1320–1331
  18. Oellerich et al., 2009 Molecular Cell Proteomics 8(7):1738-50
  19. Bekker-Jensen et al.,  2010 Nature cell biology 12(1)

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