One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

Summary

A method is described for efficient purification of twin-Strep-tagged fusion proteins and their specific complexes on modified streptavidin (Strep-Tactin) resin covalently cross-linked with Bis(sulfosuccinimidyl) suberate (BS3). The method has the advantages of fast speed, good target protein recovery and high purity, and is compatible with subsequent analysis by mass spectrometry.

Abstract

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners.

Introduction

In recent years, Strep-tag technology has become widely used in many areas of biomedical research, including proteomics and structural biology. This protein purification technology, which relies on the fusion of recombinant proteins to a short Strep-tag peptide, has matured with the advent of affinity matrices carrying Strep-Tactin, a genetically engineered variant of streptavidin with improved peptide-binding capacity.^1,2 Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, exhibit a higher affinity for Strep-Tactin matrices than those containing only a single Strep-tag, ensuring more efficient purification of the recombinant proteins and their associated binding partners. However, the higher affinity of twin-Strep-tagged proteins to Strep-Tactin also has its downside. Competitive elution of such proteins with excess biotin may be incomplete, leading to decreased target protein yield. A more efficient alternative is elution with SDS, but it leads to undesired sample contamination with Strep-Tactin released from the resin, making the assay incompatible with proteomic analysis. This paper presents a technique to overcome this limitation by first stabilizing the resin-coupled tetramer of Strep-Tactin by chemical cross-linking and then using SDS to elute twin-Strep-tagged proteins and their associated complexes from the resulting cross-linked resin. Thus, sufficient protein yield can be achieved without sample contamination with Strep-Tactin, thereby allowing further analysis by mass spectrometry.

The method is suitable for purification of any recombinant fusion protein with a surface-exposed SIII-tag³ or twin-Strep-tag (amino acid sequence WSHPQFEK(GGGS)₃ WSHPQFEK and SAWSHPQFEK(GGGS)₂ GGSAWSHPQFEK, respectively). The protein can be of animal, plant or bacterial origin and can be isolated from either total cell lysate or enriched organelle fraction. As an example, we describe here the purification of an SIII-tagged protein VPg-Pro of Potato Virus A (PVA)⁴ from the nuclear fraction of PVA-infected Nicotiana benthamiana plants. The nuclear fraction was isolated as previously described⁵, with the following modifications: cells were not treated with formaldehyde, sodium butyrate was substituted in all buffers with 5 mM sodium fluoride, complete protease inhibitor was substituted with PMSF, Triton X-100 concentration in extraction buffer #2 was lowered to 0.3% (v/v) and the nuclear pellet obtained by centrifugation through sucrose cushion (extraction buffer #3) was resuspended in 1.45 ml of pre-chilled binding buffer and rotated for 1.5 hours at 4 °C. The resulting nuclear extract containing the SIII-tagged bait protein and associated complexes (bait protein sample) was processed according to the protocol described below (see section 2).

Protocol

1. Cross-linking of Strep-Tactin Polymethacrylate Resin with Bis(sulfosuccinimidyl) Suberate (BS3)

1. Equilibrate one sealed microtube containing 2 mg of BS3 cross-linker to room temperature. CAUTION: BS3 is a hazardous substance. Wear protective gloves and goggles.
2. Resuspend Strep-Tactin polymethacrylate resin (50% suspension in 100 mM Tris-HCl, pH 8.0; 1 mM EDTA; 150 mM NaCl) by brief vigorous shaking and immediately transfer 600 µl of the suspension to a spin column using a pipette tip with the end cut off.
3. Centrifuge at 1,500 x g for 30 sec at room temperature. Discard the flow-through and add 450 µl of phosphate buffered saline (PBS) to the column.
4. Repeat the previous step 2 more times to completely replace Tris buffer with PBS and leave the resin in 430 µl of PBS adjusted to pH 8.0 after the last centrifugation.
5. Puncture the foil of the microtube with BS3 with a pipette tip containing 100 µl of ultrapure water. Dissolve the BS3 powder in water by gently pipetting up and down and immediately add 20 µl of the solution to the spin column. The final concentration of BS3 in the cross-linking reaction is ~1.2 mM.
6. Rotate the column for 30 min at room temperature. Check that the resin is mixed properly with the BS3 solution.
7. To quench the reaction, add 6 µl of 3M Tris-HCl, pH 7.5 and rotate the column for another 15 min at room temperature.
8. Centrifuge at 1,500 x g for 30 sec at room temperature. Discard the flow-through and resuspend the cross-linked resin in 450 µl of Tris buffered saline with Tween 20 (TBST). Repeat the centrifugation and wash steps two more times. At the last step, resuspend the resin in 450 µl of TBS.
9. Transfer the resin suspension to a fresh tube using a pipette tip with the end cut off. Resuspend the resin left in the column with another 450 µl of TBS and transfer to the same tube. Repeat the last step once more to ensure maximum transfer of the resin from the column to the tube.
10. Let the tube stand for 10 min at room temperature and adjust the volume to 600 µl by removing excess TBS. The resin is ready for immediate use (recommended) or can be stored at 4 °C, without freezing.

2. Binding of the Twin-Strep-tagged Bait Protein and Associated Complexes to the Cross-linked Strep-Tactin Polymethacrylate Resin

1. Centrifuge 1 ml of the bait protein sample in binding buffer at 17,000 x g for 10 min at 4 °C and transfer the supernatant to a fresh tube.
2. To minimize binding of endogenous biotinylated proteins to the Strep-Tactin resin, add avidin to a final concentration of 100 µg/ml and rotate for 15 min at 4 °C.
   1. If the cross-linked resin from step 1.10 has been stored for an extended period of time at 4 °C, collect the resin by centrifugation at 400 x g for 1 min at 4 °C, discard the supernatant and wash the resin with 1 ml of TBST. Repeat the centrifugation and wash steps two more times, first with TBST and then with TBS. Centrifuge the tube at 400 x g for 1 min at 4 °C and adjust to the original volume by removing excess TBS.
3. Resuspend the cross-linked Strep-Tactin resin by vortexing. Immediately add 50 µl of the resin suspension to the tube containing the bait protein sample using a cut pipette tip and rotate for another 30 min at 4 °C.
4. While waiting, set thermomixer to 55 °C and preheat 500 µl of elution buffer for use in step 3.1.
5. Centrifuge at 400 x g for 1 min at 4 °C. Discard the supernatant and wash the resin on a rotator for 5 min at 4 °C with 1 ml of pre-chilled wash buffer #1. Repeat the centrifugation and wash steps three times. At the last step, resuspend the resin in 250 µl of wash buffer #2.
6. Transfer the resin suspension to a fresh spin column. Resuspend the resin remaining in the tube in another 250 µl of wash buffer #2 and transfer to the same column.
7. Centrifuge at 400 x g for 3 min at 4 °C, discard the flow-through and transfer the column to a fresh dolphin-nose 2 ml tube. Proceed immediately to the elution step below.

3. Elution of Specific Protein Complexes

1. Add 150 µl of preheated elution buffer from step 2.4 to the spin column.
2. Incubate in thermomixer for 5 min at 55 °C, shaking at 1,400 rpm.
3. Centrifuge at 1,500 x g for 1 min at room temperature.
4. Discard the column and store the purified target proteins at ≤ -20 °C.

Phosphate buffered saline (PBS)
Na2HPO4	10 mM
KH2PO4	2mM
NaCl	137 mM
KCl	2.7 mM
pH adjusted to 7.4 unless stated otherwise (pH 8.0 in section 1.4)
Tris buffered saline (TBS)
Tris-HCl, pH 7.4	50 mM
NaCl	150 mM
Tris buffered saline with Tween 20 (TBST)
Tris-HCl, pH 7.4	50 mM
NaCl	150 mM
Tween 20	0.1% (v/v)
Binding buffer
Tris-HCl, pH 8.0	25 mM
NaCl	550 mM
NaF	5 mM
EDTA	0.5 mM
Glycerol	10% (v/v)
PMSF	0.1 mM
Wash buffer #1
Tris-HCl, pH 8.0	25 mM
NaCl	500 mM
NaF	5 mM
EDTA	0.4 mM
Igepal CA-630	0.2% (v/v)
Glycerol	5% (v/v)
PMSF	0.1 mM
Wash buffer #2
Tris-HCl, pH 8.0	25 mM
NaCl	150 mM
Elution buffer
Tris-HCl, pH 8.0	25 mM
SDS	1% (w/v)

Table 1. Buffers used in the present study.

Results

The purification procedure is schematically illustrated in Figure 1, together with a representation of problems associated with other existing purification methods.

Figure 1. Schematic re...

Discussion

The above protocol can be used to purify any twin-Strep-tagged bait protein of interest and its associated complexes in any suitable buffer that does not contain biotin or strong denaturants. In the current version of the protocol, binding and washing are performed under relatively stringent conditions in the presence of high salt and non-ionic detergent. Although this results in less background, fragile protein complexes may dissociate under these conditions. To preserve such lower affinity complexes, salt concentration...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bis(sulfosuccinimidyl) suberate (BS3), No-Weigh format, 8 x 2 mg	Pierce/Thermo Scientific	21585	www.fishersci.com CAUTION: Hazardous substance. Causes serious respiratory, skin and eye irritation. Wear protective gloves and eye protection.
Strep-Tactin MacroPrep resin (50% suspension)	IBA	2-1505	www.iba-lifesciences.com
Spin-X centrifuge tube filter, cellulose acetate membrane, pore size 0.45 μm, non-sterile	Costar (Corning)	8163	www.corning.com/lifesciences/
Dolphin-nose tubes	Costar (Corning)	3213	www.corning.com/lifesciences/
Avidin	IBA	2-0204	www.iba-lifesciences.com