Newsletter Issue 7 One-STrEP Analysis of Protein:Protein-Interactions

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1 Newsletter Issue 7 One-STrEP Analysis of Protein:Protein-Interactions

2 Strep-tag and One-STrEP-tag PPI Analysis with the co-precipitation/ mass spectrometry approach 3 Background The Strep-tag was originally selected from a random library for specific streptavidin binding activity thereby enabling purification of corresponding fusion proteins on streptavidin affinity columns. Binding reversibly to the same pocket where the natural ligand D-biotin is complexed, elution of the bound recombinant protein is effected by competition with D-biotin. Analogues with less strong affinity, such as D- desthiobiotin, can be used as well thus facilitating repeated use of the column. The system was systematically optimized over the years 1, including development of the optimized Strep-tag II for N- or C-terminal as well as protein internal fusion and engineering of a streptavidin variant with improved binding capacity, dubbed Strep- Tactin. Properties A particular benefit of Strep-tag II is that it has a neutral amino acid composition and that it does not hamper protein folding or secretion, nor does it interfere with protein function. Strep-tag enables purification of recombinant proteins to over 99% purity in a single step from crude lysates. The extraordinary purification factors are based on i) very low tendency of Strep-Tactin to bind other proteins non-specifically, ii) highly specifc Strep-tag II:Strep-Tactin interaction and iii) specific competitive elution with minute amounts of desthiobiotin in the physiological wash buffer. Moreover, extreme stability of Strep-Tactin is the basis of robust affinity resins. excise preys and analyse by mass spectrometry 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 are excised and identified by mass spectrometry. 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 determined 2. These properties predestine the Strep-tag :Strep-Tactin system for PPI analysis according to Rigaut et al., 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. 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. Strep-tag II: SAWSHPQFEK One-STrEP-tag: SAWSHPQFEK(GGGS)2GGSAWSHPQFEK References: 1. Schmidt & Skerra, 2007, Nature Protocols 2: Ostermeier et al., 1997, PNAS 94: Rigaut et al., 1999, Nature Biotech. 17: Juntilla et al., 2005, Proteomics 5: Johansen et al., 2008, J Cell Sci 121: Groth et al., 2007, Science 318: Schaffitzel & Ban, 2007, J Struct Biol 158: Morita et al., 2007, EMBO J. 26: Morita et al., 2007, Cell Host & Microbe 2: Jarchow et al., 2008, Proteomics, submitted 11. Gloeckner et al., 2007, Proteomics 7, Herzberg et al., 2007, Proteomics 7,

3 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 TM 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 eukaryotes 4,5,6,7,8,9 and prokaryotes 10. 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 discriminate 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 published 11, 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-STrEP TM 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 Experiment 12 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 prokaryotes 12 only but its adaptation to mammalian systems is under way.

4 One-STrEP TM 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. Please note that weakly-binding prey 5 and non-specifically binding protein 6 may be co-purified. 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. Product information see last pagennn One-STrEP Set for mammalian cells cat. no One-STrEP Set for E. coli cells cat. no 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 ) Please note that non-specifically binding protein 6 but also weaklybinding prey 5 may be removed during the second purification step. Disadvantages and Risks Purification with two columns makes method more time-consuming and complex Risk of loosing weak binding partners during second purification step Product information see last pagennn One-TAP Set for mammalian cells cat. no One-TAP Set for E. coli cells cat. no

5 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 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. Ca 2+ ) binding FLAG Product information see last pagennn Two-TAP Set for mammalian cells cat. no Two-TAP Set for E. coli cells cat. no Please note that weakly-binding prey 5 and non-specifically binding protein 6 may be lost during the second purification step, while a potential non-specific FLAGbinding protein 7 may remain attached. 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 Product information see last pagennn SPINE Set for E. coli cells cat. no Please note that stringent wash conditions are possible without removing weakly-binding prey 5, which is cross-linked, while non-specifically binding protein 6 may be washed off.

6 StarGate for Bait Cloning This novel cloning system is the perfect tool for efficient screening and fast identification of the optimal tag for PPI investigation with a given bait. Once the bait protein is cloned into the Donor Vector, a large selection of Acceptor Vectors for its expression with different tag arrangements is available. For PPI analysis we recommend to start with Acceptor Vectors equipped with the following features: One-STrEP-tag at N- and C-terminus (for One-STrEP, One-TAP, SPINE) One-STrEP/ FLAG -tag double tag, N- and C-terminal (for Two-TAP) Strep-tag II at N- and C-terminus (for SPINE) The list of Acceptor Vectors below gives a comprehensive overview of all options currently available (cat. nos. of recommended vectors are high-lighted in blue). The recommended Acceptor Vectors are included in the PPI Sets (see table on last page) but can also be purchased separately. For detailed information on the StarGate TAGnology, please visit our website at StarGate Acceptor Vector Overview

7 Products for PPI For each of the described methods we offer a complete Set (for mammalian cells or E. coli) which includes all relevant products from cloning of the bait via StarGate (Cloning Kit) to purification of the protein complex (Purification Kit) (see table on last page). The kits and also single products can be purchased separately. Both a comprehensive StarGate Manual (PR26) and a PPI Manual (PR27) are delivered with the Kits and Sets. Where to order order@iba-go.com for more options please see last page. Non-covalent prey capture One-STrEP products The components of the One-STrEP Set for mammalian cells and E. coli are listed in the brown column of the table on the last page. Please note that IBA s former kits for mammalian cells, the One-STrEP Starter Kit (cat. no ) and the One-STrEP Follow Up Kit (cat. no ), are still available; these kits include the IBA Classic Vectors pexpr-iba103 and pexpr-iba105, respectively, and are thus designed for IBA s classic cloning procedure. For cloning via StarGate please use the One-STrEP Sets as described here (see table on last page). One-TAP products The components of the One-TAP Sets for mammalian cells and E. coli are listed in the yellow column of the table on the last page. For the first purification step the same products are required as listed for the One- STrEP procedure; for the second purification step additionally a StrepMAB-Classic MacroPrep column and the Strep-tag II peptide for elution are necessary. Two-TAP products The components of the Two-TAP Sets for mammalian cells and E. coli are listed in the green column of the table on the last page. For the first purification step (via the One-STrEP-tag) the same products are required as listed for the One-STrEP procedure; for the second purification step (via the FLAG tag) an anti-flag M2 agarose column and a FLAG octapeptide for elution are necessary. These FLAG -products are not offered by IBA but can be purchased from Sigma. One-STrEP TM One-TAP Two-TAP Covalent prey capture SPINE products The components of the SPINE Set for E. coli are listed in the blue column of the table on the last page. Cross-linking of bait and preys in vivo is achieved with p-formaldehyde which is not offered by IBA but has to be purchased elsewhere (e.g., Roth, #0335.1). For the subsequent purification of the protein complex via the Strep-tag II or the One-STrEPtag the same products are required as listed for the One-STrEP procedure. SPINE Further IBA Products IBA offers many further reagents for the analysis of Strep-tag II and One-STrEP-tag fusion proteins like the nearly irreversibly binding StrepMAB-Immo (monoclonal antibody) for directed and controlled immobilization of baits or protein complexes on Biacore chips or microplates. For more information please refer to Products featuring CMV promoter, GSTtag, 6x Histidine-tag, One-STrEP tag TM, StarGate, Strep-Tactin, Strep-tag, T7 promoter, tet promoter are covered by patents and patent applications and Limited Use label Licenses. Please refer to for the corresponding statements. Products are for research use only.

8 Overview of Sets, Kits and Products for PPI