1 Introduction

The application of systematic TAP-tag approaches had an enormous impact on our understanding of protein complexes and their function within cells. The primal TAP-tag strategy was developed and applied in yeast [1] and allowed the description of a tremendous amount of protein complexes and their members [2]; however, in several other expression systems, this tag was hardly functional or even failed [3, 4]. Therefore, a variety of other TAP-tags were developed to overcome these limitations (e.g., ref. 5–7).

Here we describe the usage of the synaptobrevin Avi purification-tag (SnAvi-tag)—our version of a TAP-tag [8]. Similar to other TAP-tags, the SnAvi-tag is composed of several genetically encoded modules (Fig. 1) which enable its application in different experimental contexts. The most important of those modules is the AviTag [9, 10], a short peptide sequence which is specifically and exclusively recognized by the E. coli biotin holoenzyme synthetase BirA. Only when BirA is expressed in the same cells as the SnAvi-tagged protein, the tag structure is biotinylated and thus activated for protein purification using (strept-)avidin coupled affinity matrixes. This feature is especially interesting for the application in multicellular organisms, since it enables the expression of an endogenous gene under the regulation of its own promoter and the purification of the tagged-protein from a subset of cells which is defined by a selective co-expression of BirA. An additional advantage of the application of the AviTag for the first protein enrichment step in the sequential purification procedure is determined by the femtomolar dissociation constant of (strept-)avidin to biotin [11] which enables the enrichment of low-abundant proteins and stringent washing conditions to remove contaminants.

Fig. 1
figure 1

Schematic view of a SnAvi-tagged target protein. The target protein is fused to the SnAvi-tag that is composed of two epitopes for the SB1 antibody, two recognition motifs for the TEV protease, the coding sequence for a variant of the green fluorescent protein (EGFP), and the AviTag. In eukaryotic cells the AviTag is specifically and exclusively recognized and biotinylated by the E. coli biotin holoenzyme synthetase BirA that we express as fusion to the mCherry fluorophore

Full size image

In order to detect the cellular or subcellular localization of the SnAvi-tagged proteins, the peptide sequence of the green fluorescent protein (GFP, [12]) was added. Two TEV-protease recognition motifs are also included in the tag structure. They are useful to specifically elute tagged proteins from the (strept-)avidin coupled affinity matrix after the first purification step [13], since the GFP-AviTag fusion and contaminants unspecifically bound to it can be removed by protease cleavage. To allow the concentration of the eluted protein complexes, two epitopes for the SB1 peptide antibody were introduced into the SnAvi-tag structure for the second step of the tandem purification strategy. The epitope of this antibody is derived from the synaptobrevin gene 1 (snb-1) of C. elegans and is detected in synthetic protein fusions in C. elegans as well as in cultivated mammalian cell lines with a high specificity. Thus, the SnAvi-tag is applicable for sequential protein purification in a broad variety of eukaryotic cells.

2 Materials

Prepare all solutions with ultrapure water (resistance > 18 MΩ) and reagents of analytical grade. Sterilize all solutions either by filtration or autoclaving. If not otherwise specified, store all buffers at room temperature.

2.1 Growth, Transfection and Lysis of Cultivated Mammalian Cells

  1. 1.

    Choose cell line for the expression of the SnAvi-tagged protein fusion.

  2. 2.

    Appropriate growth medium for the cell line to be transfected (see Note 1 ).

  3. 3.

    PBS: 8 g/l NaCl, 0.2 g/l KCl, 1.78 g/l Na2HPO4 × H2O, 0.24 g/l KH2PO4, adjust pH to 7.4 (see Note 2 ).

  4. 4.

    1 mg/ml 25 kDa linear polyethylenimine (PEI, see Note 3 ); pH 7.2.

  5. 5.

    1 M d-biotin (Sigma) stock solution in the appropriate cell culture medium.

  6. 6.

    Cell lysis buffer: 50 mM Tris (pH 7.4), 1 % Triton X-100, 137.5 mM NaCl, 1 % glycerol, 1 mM NaO4Va, 0.5 mM EDTA, Protease Inhibitor Cocktail (Roche Applied Sciences).

2.2 Work with C. elegans

  1. 1.

    S basal buffer: 5.8 g NaCl, 50 ml 1 M KH2PO4 (pH 6.0; see Note 4 ), 950 ml H2O, 1 ml Cholesterol stock solution (5 mg/ml in 95 % EtOH absolute; see Note 5 ).

  2. 2.

    100× metal solution (500 ml): 0.346 g FeSO4 × 7 H2O, 0.098 g MnCl2 × 4 H2O, 0.144 g ZnSO4 × 7 H2O, 0.012 g CuSO4 × 5 H2O, 0.930 g Na2 EDTA.

  3. 3.

    Nystatin stock solution: dissolve 1 g Nystatin (Sigma) in 50 ml EtOH (p. A.) and 50 ml 7.5 M NH4Acetate.

  4. 4.

    Nematode growth medium (NGM)—plates : 3 g NaCl, 17 g agar, 2.5 g peptone (Bacto), fill up to 950 ml with H2O; after autoclaving add 5 μg/ml Cholesterol, 1 mM CaCl2, 1 mM MgSO4, 5 mM KH2PO4 (pH 6.0), 20 μg Nystatin, fill up to 1 l.

  5. 5.

    Liquid culture medium: supplement 50 ml S basal buffer with 150 μl 1 M MgSO4, 150 μl 1 M CaCl2, 0.5 ml 100× metal solution, 0.5 ml 1 M citrate (pH 6.0; adjust with concentrated KOH), 0.5 ml 100× Penicillin/Streptomycin stock solution (Gibco), 2 ml pelletized HB101 bacteria.

  6. 6.

    M9 buffer: 3 g/l KH2PO4, 6 g/l Na2HPO4, 5 g/l NaCl, adjust pH to 6.0 and autoclave; autoclave separately a 1 M MgSO4 stock solution; add 1 ml MgSO4 solution per each l of the autoclaved buffer components.

  7. 7.

    60 % ice-cold sucrose solution (w/w).

2.3 Tandem Affinity Purification

  1. 1.

    IPP150 lysis and washing buffer: 10 mM Tris HCl (pH 8), 150 mM NaCl, 0.1 % NP-40, 3× concentrated protease inhibitor cocktail (Roche Applied Sciences; see Note 6 ).

  2. 2.

    TetraLink Avidin Resin (Promega; see Note 7 ).

  3. 3.

    TEV cleavage buffer: 0.5 mM EDTA, 1 mM DTT, 150 mM NaCl, 50 mM Tris (pH 8, see Note 8 ).

  4. 4.

    AcTEV protease (Invitrogen).

  5. 5.

    Spin Cups—cellulose acetate filter (0.45 μm pore size; Thermo Scientific).

  6. 6.

    Crosslink IP kit (Thermo Scientific).

  7. 7.

    French Press (see Note 9 ).

  8. 8.

    Modified Laemmli buffer (6×): 300 mM Tris–HCl (pH 6.8), 12 mM EDTA, 6 % SDS (w/v), 30 % (v/v) glycerol.

  9. 9.

    Gels and buffers for sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE).

2.4 Antibodies and Conjugates

  1. 1.

    High Sensitivity Streptavidin coupled to horseradish peroxidase (HRP; Thermo Scientific) in combination with enhanced chemiluminescence substrates for the detection of biotinylated SnAvi-tagged proteins on western blot membranes (see Note 10 ).

  2. 2.

    Purified SB1 antibody recognizing the SNB-1 epitope in the SnAvi-tag (see Note 11 ) is used for immunoprecipitation or detection in western blot (see Note 12 ).

3 Methods

3.1 Expression of SnAvi-Tagged Proteins in Cultivated Mammalian Cells

  1. 1.

    Grow HEK293T or HeLa cells in the respective growth medium until they are about 80–90 % confluent.

  2. 2.

    Supplement d-biotin (100 μM final concentration) to the growth medium of the cells to increase the biotinylation level of SnAvi-tagged proteins 1 h before transfection.

  3. 3.

    Use 110 ng of the birA (see Note 13 ) encoding plasmid pBY2892 per cm2 plate surface and 200–500 ng of the SnAvi-tag encoding plasmid for the co-transfection of adherently growing cells (see Note 14 ). Incubate in 200 μl medium w/o serum or antibiotics for 5 min. In parallel, incubate an appropriate PEI volume (ratio PEI volume to DNA mass: 3 μl PEI per μg plasmid DNA) in 200 μl medium w/o serum or antibiotics for 5 min.

  4. 4.

    Mix the DNA solution with the PEI solution. Incubate for another 20 min.

  5. 5.

    Pipet the DNA/PEI mixture dropwise to your cells.

  6. 6.

    Optionally: Exchange the growth medium of the cells 3 h after the DNA/PEI mixture was added.

  7. 7.

    Incubate cells for 24–48 h at 37 °C.

3.2 Preparation of Cell Lysates from SnAvi-Transfected Cells

  1. 1.

    Aspirate growth medium from the cells.

  2. 2.

    Carefully wash the cells once with ice-cold PBS.

  3. 3.

    Aspirate the PBS.

  4. 4.

    Add 16.5 μl of ice-cold cell lysis buffer per cm2 growth surface.

  5. 5.

    Incubate on ice for 15–30 min.

  6. 6.

    Scrape cells from the cell culture dish. Transfer them into a polypropylene (PP) tube.

  7. 7.

    Clear the lysates by centrifugation (about 15,000 rcf, 4 °C, 15 min).

  8. 8.

    Transfer the supernatant to a fresh tube.

  9. 9.

    Proceed with “Tandem affinity purification of SnAvi-tagged proteins”

3.3 Expression of SnAvi-Tagged Proteins in C. elegans

  1. 1.

    Transform C. elegans either by microinjection transformation [14] or by biolistic transformation [15, 16], see Note 15 ) with the desired fusion of a gene and the SnAvi-tag coding sequence (see Note 16 ). Moreover, strains expressing the birA coding sequence must also be generated (see Note 18 ). When microinjection transformation is applied, the DNA for both can be co-transformed. If strains are made which carry only one of the two required transgenes, both traits have to be combined by genetic crossing in an additional step (see Note 17 ) before the SnAvi-Tag can be used.

  2. 2.

    Add 50 ml of liquid culture medium to a 250 ml Erlenmeyer flask (see Note 18 ).

  3. 3.

    Add the transgenic worms grown on ten NGM plates (10 cm diameter) seeded with the E. coli strain OP50 (wash animals from plates; collect the worms by centrifugation with 200 rcf, 4 °C, 1 min).

  4. 4.

    Incubate the Erlenmeyer flask in a shaking incubator (20 °C, 130 rpm) for several days until you have enough worm mass (usually 4–8 day).

  5. 5.

    Check every day if animals are starving. If dauer larvae are observed feed the animals with more HB101 bacteria (add 1–2 ml of bacterial pellet).

  6. 6.

    Pelletize the animals (200 rcf, 4 °C, 1 min).

  7. 7.

    Wash the worm pellet with ice-cold M9 buffer.

  8. 8.

    To separate living animals from dirt and dead worms transfer them to 50 ml PP-tubes.

  9. 9.

    Add 20 ml ice-cold M9 buffer.

  10. 10.

    Add 20 ml ice-cold 60 % sucrose solution w/w (40 g sucrose per 60 g water).

  11. 11.

    Mix shortly. Proceed instantly to the next step.

  12. 12.

    The sucrose floating of vital worms is established during centrifugation (860 rcf, 5 min, 4 °C). Carefully aspirate the floating worms in the upper phase with the help of a Pasteur pipette and transfer them to a fresh 50 ml PP tube.

  13. 13.

    Fill with ice-cold M9 buffer up to 50 ml.

  14. 14.

    Pelletize the worms.

  15. 15.

    Repeat the steps 13 and 14 twice.

  16. 16.

    Aspirate the supernatant.

  17. 17.

    Either prepare worm lysates immediately or store the pellets at –80 °C.

3.4 Lysate Preparation from C. elegans

  1. 1.

    Combine 0.5 ml of worm pellet with 4.5 ml of IPP150 buffer.

  2. 2.

    Press the animals through a precooled French pressure cell (see Note 3 ) that is appropriate for the required volume. Apply 8,000 psi pressure.

  3. 3.

    Repeat this step twice.

  4. 4.

    Proceed with “Tandem affinity purification of SnAvi-tagged proteins”.

3.5 Tandem Affinity Purification of SnAvi-Tagged Proteins

All steps are carried out at 4 °C if not stated otherwise. All centrifugation steps with TetraLink material are performed with 100 rcf, 4 °C, 1 min. All centrifugation steps with the SB1-coupled Protein A/G material are conducted with 2,000 rcf, 4 °C, 1 min. The cross-link of the SB1 antibody has to be accomplished with the “Crosslink IP kit” (Thermo Scientific) in advance according to the supplier’s protocol. Protein A/G affinity matrix carrying the cross-linked antibody can be stored for several days.

  1. 1.

    Transfer TetraLink beads to a fresh PP-tube. Allow to settle. Use 80 μl affinity matrix per ml lysate (see Notes 19 and 20 ).

  2. 2.

    Wash TetraLink beads twice with IPP50 buffer (C. elegans lysates) or cell lysis buffer (lysates from mammalian cultivated cells).

  3. 3.

    Aspirate most of the washing buffer but leave approximately the same volume of buffer as the volume of affinity matrix.

  4. 4.

    Add your lysate.

  5. 5.

    Incubate on an “end over end rotator” at 4 °C for 30 min.

  6. 6.

    Wash 5× with IPP50 buffer (C. elegans lysates) or cell lysis buffer (lysates from mammalian cultivated cells).

  7. 7.

    Wash twice with TEV cleavage buffer.

  8. 8.

    After the last washing step, aspirate most of the buffer without drying the beads.

  9. 9.

    Add 0.5 U/μl AcTEV protease to a volume of TEV-cleavage buffer that is 3× higher than the volume of the affinity matrix material.

  10. 10.

    Incubate at 16 °C for 2 h (see Note 21 ).

  11. 11.

    Transfer the supernatant and the beads to spin cup—cellulose acetate filters.

  12. 12.

    Separate the supernatant from the affinity matrix material by centrifugation (7,000 rcf, 4 °C, 2 min, see Note 22 ).

  13. 13.

    Add settled Protein A/G affinity matrix which was cross-linked with the SB1 antibody before. Use 5 μl affinity matrix per ml lysate (see Note 23 ).

  14. 14.

    Incubate on an “end over end rotator” at 4 °C over-night.

  15. 15.

    Wash 3× with 500 μl TEV cleavage buffer.

  16. 16.

    Aspirate the TEV cleavage buffer.

  17. 17.

    Add sample buffer (e.g., per 10 μl affinity matrix add 15 μl 2× concentrated modified Laemmli buffer).

  18. 18.

    Elute the bound protein and its complex partners by incubation at 95 °C for 3–5 min.

  19. 19.

    Separate proteins by SDS-PAGE (see Notes 24 – 26 ).

4 Notes

  1. 1.

    We normally work with HEK293T or HeLa cells. Both are grown in MEM medium (PAA Laboratories) supplemented with 10 % fetal bovine serum (PAA), 50 U/ml Penicillin, 50 μg/ml Streptomycin (100 × stock, Gibco). The cells are cultivated with 5 % CO2 at 37 °C.

  2. 2.

    We normally prepare a 10× PBS stock solution.

  3. 3.

    Heat the aqueous solution at 80 °C until the PEI (Polysciences Inc.) is completely dissolved.

  4. 4.

    Dissolve 136.1 g KH2PO4 in 800 ml H2O; use concentrated KOH to adjust pH to 6.0; fill up to 1,000 ml.

  5. 5.

    Do not autoclave the cholesterol stock solution!

  6. 6.

    Since during the lysis of C. elegans a huge number of intestinal proteases are released, we always use a 3 times higher concentration of protease inhibitors as recommended by the company.

  7. 7.

    We tested several similar products from other companies. In our hands this affinity matrix had the best price/efficiency relation.

  8. 8.

    The TEV-protease is not sensitive for the Roche protease inhibitor cocktail when it is applied 1× concentrated. Therefore, this protease inhibitor cocktail can optionally be added.

  9. 9.

    Instead of using the French press, worm lysates can also be prepared by grinding the animals in liquid nitrogen or by sonication according to standard protocols. In the former case, the protein powder is transferred to polypropylene tubes before the IPP150 lysis buffer is added. In the latter case, the IPP150 lysis buffer is directly added before sonication. In both cases clear the lysates by centrifugation and use the supernatant for the following steps (16,100 rcf, 4 °C, 15 min).

  10. 10.

    Alternatively, we use Alexa680 coupled streptavidin (Invitrogen) for the detection of biotinylated SnAvi-tagged proteins with the Odyssey Scanner (LI-COR). This strategy has the advantage that a second detection can be done in parallel. We used this strategy for example to improve the efficiency of TEV-protease cleavage using the SB1 antibody for the detection of the SnAvi-tag after cleavage and Alexa 680 streptavidin for the detection of the unprocessed form.

  11. 11.

    The SB1 antibody developed by Michael Nonet and Gayla Hadwiger was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. It is publicly available. The peptide recognized by the antibody has the sequence PRPSNKRLQQ [8] and is specifically recognized in folded as well as denaturated proteins [8, 17]. We produced the antibody in CD hybridoma medium (Gibco) or enriched the antibody from serum supplemented DMEM medium with HPLC using HiTrap™ Protein G HP Columns (GE Healthcare). In the latter case, 0.5 M Na3Citrate (pH 8.2) was used for washing, and 0.1 M Na3Citrate (pH 3) was used for elution. 50 μl 1 M Tris (pH 9.5) per ml eluate were added for neutralization. A concentration of 4.6 μg/ml SB1 antibody was used for detection on western blot.

  12. 12.

    Alternatively to the SB1 antibody, commercially available α-GFP antibodies are useful to detect SnAvi-tagged proteins.

  13. 13.

    Alternatively, to the in vivo biotinylation of SnAvi-tagged proteins in C. elegans or in mammalian cell culture, SnAvi-tagged proteins can also be biotinylated in E. coli by the endogenous BirA protein, e.g., in the BL21 E. coli strain. However, we failed so far to express any protein in the AVB100 strain in which recombinant birA expression is induced in addition to the endogenous birA. Moreover, in vitro biotinylation of the SnAvi-tag is possible using the BirA enzyme from Avidity (Aurora, CO, USA).

  14. 14.

    Dependent on the gene-of-interest, different concentrations of plasmids encoding SnAvi-tagged proteins should be tested to identify the best expression conditions.

  15. 15.

    We prefer biolistically transformed rescued DP38 (unc-119(ed3)) lines, since stably integrated transgenes can be obtained with this method [15, 16].

  16. 16.

    SnAvi encoding vectors are available from Addgene (www.addgene.org). They are compatible either for expression in C. elegans (pBY2946), for expression exclusively in mammalian cells (pBY2727), or for expression in bacteria, insect and mammalian cells (pBY2807, pBY2887). To activate SnAvi-tagged proteins by biotinylation, the E. coli biotin holoenzyme synthetase gene birA has to be co-expressed in the same cells. The cDNA of this gene is available from Addgene as a fusion to the mCherry cDNA sequence (pBY2982). This vector can also be adapted for birA-mCherry expression in C. elegans by inserting C. elegans specific promoters 5′ to the start ATG of the birA-mCherry coding sequence.

  17. 17.

    For mass spectrometry analysis, a strain that carries only a SnAvi-tagged transgene but does not express BirA could be used as negative control in SILAC experiments [18].

  18. 18.

    We tried to grow high numbers of worms expressing SnAvi-tagged proteins on egg plates. However, even though the tandem-affinity purification should bias for SnAvi-tagged proteins and their complex partners, chicken avidin was still co-purified, reducing the sensitivity of mass spectrometry (MS) runs. We suggest therefore to exclusively use animals grown in liquid culture for the enrichment of SnAvi-tagged proteins followed by MS measurements. If the enriched proteins are analyzed with different methods, it might be useful to grow the worms on egg plates, since they are easier to handle than liquid worm culture. Composition of egg plates: prepare NGM plates (10 cm diameter, 30 ml volume) with agarose instead of agar; use one sterilized chicken egg per six plates (see Note 25 ), fill up with LB to 40 ml total volume, incubate at 60 °C for 1 h to inactivate the lysozyme in the egg, cool down to RT, add 5 ml bacterial suspension (best use the E. coli strain OP50), equally distribute to the plates, and allow to settle over-night. The next morning carefully aspirate the supernatant from the plates. Plates can be stored for several weeks at 4 °C.

  19. 19.

    You should be able to wash the affinity matrix in a washing buffer volume that is at least 30× more than the volume of your affinity matrix. The size of the PP-tube should be chosen accordingly. We normally use 15 ml PP-tubes for 0.4 ml settled affinity matrix and 5 ml lysate.

  20. 20.

    Although the SnAvi-Tag benefits from the femtomolar dissociation constant of its first affinity stage, one has to consider that endogenously biotinylated proteins which are produced in all species will occupy binding capacity on the streptavidin affinity matrix.

  21. 21.

    Dependent on the protein fused to the SnAvi-tag, other conditions (time, temperature, pH, varying AcTEV concentrations, etc.) for the AcTEV protease digestion can be tested.

  22. 22.

    In rare cases, the affinity matrix clogs the filter. In this case resuspend the material again in the residual buffer and centrifuge again or transfer the buffer/affinity matrix mixture to a fresh filter.

  23. 23.

    We also used BrCN Sepharose for cross-linking the antibody to the affinity matrix. This increased the number of detected peptides in mass spectrometry but also the number of contaminants. Nevertheless it could be considered to covalently bind the SB1 antibody.

  24. 24.

    After Coomassie Blue staining, we either cut specific bands from the SDS-PAGE for mass spectrometry or we ran the gel only shortly and cut all eluted proteins within a single band from the SDS-PAGE.

  25. 25.

    For trouble shooting in general and especially for the establishment of new SnAvi-Tag purification projects, it is very helpful to monitor the performance of the individual steps on a western blot. The amount of tagged BirA-biotinylated protein can be compared to the natively biotinylated proteins by probing a cell- or worm lysate with labeled streptavidin. After the first affinity purification, the amount of remaining tagged protein can be analyzed in the waste supernatant, and the loading of streptavidin beads can be tested with an anti-GFP-antibody. After TEV-cleavage the amount of remaining non-cleaved tagged protein should be tested by probing the eluate and the waste streptavidin matrix with the SB1 antibody. This is important for the optimization of TEV-protease cleavage conditions. For the second affinity step, the remaining protein can be tested in the waste-supernatant of the SB1-antibody pull-down.

  26. 26.

    Several SnAvi-tagged proteins degradation bands were detectable on western blots. However, in the first purification step using the Avidin coupled affinity matrix the full length protein is enriched preferentially.

  27. 27.

    Sterilize the shell of the chicken eggs in an aqueous NaOCl bath (approximately 0.12 %). Afterwards, wash twice in H2O for 10 min.