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....

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 sal....

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