Protein:Protein Interaction Analysis



Strep-tag® and One-STrEP-tag for PPI Analysis

Protein:protein-interactions (PPI) govern almost all important processes in living organisms. Thus, their rapid and accurate determination and investigation is a major challenge in life sciences. IBA provides optimal solutions with its different determination systems for protein:protein-interaction analysis.

The One-STrEP™ system (one-step purification with One-STrEP-tag on Strep-Tactin®) is recommended for getting started. It needs one tag and one purification step only and is validated for eukaryotes4,5,6,7,8,9 and prokaryotes10. Due to its excellent performance, this method yields a favorable signal-to-noise ratio in most cases. Mild elution and fast washing allow the isolation of even weekly interacting preys.

In case the One-STrEP system provides suboptimal data the "One-TAP" system (One-tag Tandem Affinity Purification with One-STrEP-tag on Strep-Tactin® and StrepMAB-Classic) extends the options of the One-STrEP system since it adds a second independent purification step yet with the same tag. Two different purification steps may better discriminat specific from non-specific binding but bear the risk of loosing weakly interacting partners.

Two different tags increase the risk of non-specific binding or interference with the native conformation of the bait necessary for an effective binding of associated proteins. Although a successful approach has already been published11, we recommend the "Two-TAP" system (Two-tag Tandem Affinity Purification with One-STrEP-tag on Strep-Tactin® and FLAG®-tag on M2 mAb) only as an option in case of unsatisfying data with the One-STrEPTM or "One-TAP" approach and not as first choice starting point.

In addition to these non-covalent capture methods of potential preys, SPINE (Strep-Protein Interaction Experiment12 with Strep-tag®II) adds the possibility to covalently link the preys to its bait by formaldehyde cross-linking. This linkage is achieved in the living organism enabling a time resolved snapshot of interacting proteins. SPINE is currently validated in prokaryotes12 only but its adaptation to mammalian systems is under way.
  Non-covalent prey capture Advantages Disadvantages
Recommended method
to start		 One-STrEP
one tag
one column
  • only one tag
  • even weakly interacting
    preys are isolated in one step
  • in some cases background due
    to non-specific proteins
    may occur

Recommended in case
of background
with One-STrEP method		 One-TAP
one tag
two columns
  • two purification steps with
    only one tag increase
    signal:noise ratio
  • recommended for high-affinity PPI
  • risk of loosing weak
    binding partners

Alternative option only
if One-TAP performs
insufficiently		 Two-TAP
two tags
two columns
  • improved purification procedure
    for FLAG-users with two tags
  • improvement of original TAP
    procedure
  • risk of increased non-specific
    binding
  • risk of interfering with native
    conformation of bait and thus
  • risk of impaired binding of preys

  Covalent prey capture
Reversible cross-linking
in vivo with formaldehyde		 SPINE
one tag
one column
formaldehyde
  • only one tag
  • time-resolved map of
    interacting proteins possible
  • validated only for
    prokaryotic
    expression systems


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. Jarchow et al., 2008, Proteomics, submitted
  11. Gloeckner et al. (2007) Proteomics 7, 4228-4234
  12. Herzberg et al. (2007) Proteomics 7, 4032-4035

top One-STrEP™

The One-STrEP system isolates protein complexes by a single affinity purification step on Strep-Tactin® Superflow®, including short washing only, thereby enabling co-purification of weakly associated preys. Physiological buffers are used throughout the purification process and elution is performed with minute concentrations of biotin in the same buffer.



Advantages

  • Only one tag, only one purification step
  • Fast and easy purification conserves even weak protein:protein-interactions
  • Good signal-to-noise ratio
  • Mild elution conditions conserve functional structures of protein complexes
  • Convenient and highly flexible bait cloning procedure available (StarGate®)

Disadvantages and Risks

  • In some cases background due to non-specifically binding proteins may occur




Order information
see also "how to order"

Product information see products / order cat. no.
One-STrEP Set for mammalian cells 2-1121-001
One-STrEP Set for E. coli cells 2-1121-002


 

The One-STrEP procedure



Cells are transfected with the plasmid coding for the bait protein fused to the One-STrEP-tag. After an appropriate expression period, cells are lysed and the bait together with bound proteins is isolated using a Strep-Tactin column. Proteins are resolved on a SDS-gel and specifically bound proteins e.g. proteins not appearing in the mock control can be identified. This is done by either excising the protein bands from the gel and subsequent mass spectrometric analysis or, if interaction partners are known or expected, by Western blot using specific antibodies.


top One-TAP

In the first step, the One-STrEP-tag bait protein and its binding partners are isolated as described above and further purified in a second step on a column with the One-STrEP-tag monoclonal antibody StrepMAB-Classic immobilized to MacroPrep®. Both steps are substantially different with respect to the resin and affinity receptor. Mild elution with biotin in step 1 and with the Strep-tag®II peptide in step 2 allow the isolation of the protein complex at highest purity under physiological conditions.



Advantages

  • Only one tag minimizes influence on bait protein
  • Two independent purification steps increase signal-to-noise ratio
  • Discrimination of non-specific and weak binding partners possible by comparison of different methods (One-STrEP and One-TAP)
  • Well-suited for strong protein:protein interactions
  • Convenient and highly flexible bait cloning procedure available (StarGate®)

Disadvantages and Risks

  • Purification with two columns makes method more time-consuming and complex
  • Risk of loosing weak binding partners during second purification step




Order information
see also "how to order"

Product information see products / order cat. no.
One-TAP Set for mammalian cells 2-1122-001
One-TAP Set for E. coli cells 2-1122-002




top Two-TAP

As the highly charged metal ion binding FLAG®-tag bears the risk of non-specific interactions, – in fact, metal ions are often part of active centers of enzymes and involved in signalling cascades - this system is recommended only if the performance of One-TAP for a given protein complex is insufficient. In the first step, One-STrEP/FLAG® double-tagged proteins are eluted from Strep-Tactin® Superflow® under native conditions with biotin, in the second step elution from anti-FLAG M2 agarose is performed with the FLAG octapeptide.



Advantages

  • Improved purification procedure for FLAG tag users
  • Two independent purification steps
  • Convenient and highly flexible bait cloning procedure available (StarGate®)

Disadvantages and Risks

  • Purification with two tags and two columns makes method more time-consuming and complex
  • Risk of increased non-specific binding
  • Risk of interference with cellular processes and the native conformation of the bait due to metal ion (e.g. Ca2+ ) binding FLAG




Order information
see also "how to order"

Product information see products / order cat. no.
Two-TAP Set for mammalian cells 2-1123-001
Two-TAP Set for E. coli cells 2-1123-002



top SPINE

In this method the advantages of the One-STrEP purification system are combined with those of reversible in vivo protein cross-linking. Formaldehyde links proteins which are in close contact to each other thereby fixing the cellular protein:protein-interaction status at a given point of time. Stabilized protein complexes are purified on Strep-Tactin® and then resolved into components by short heating for further analysis.



Advantages

  • Only one tag (Strep-tag® II)
  • Preys are covalently linked to the bait and weakly associated preys cannot escape detection
  • High-stringency wash procedure reduces background
  • Fast and easy one-step purification
  • Time-resolved analysis of binding processes possible
  • Convenient and highly flexible bait cloning procedure available (StarGate®)

Disadvantages and Risks

  • At present validated for Strep-tag® II and prokaryotic expression systems only




Order information
see also "how to order"

Product information see products / order cat. no.
SPINE Set for E. coli cells 2-1124-002




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