We gratefully acknowledge the technical support of Sini Miettinen, Minna P&#246;ll&#228;nen and Taru Rautavesi. We thank Helka Nurkkala for providing HEK 293 cells expressing twin-Strep-tagged GFP and Pekka Evij&#228;rvi for providing sound recording equipment. This work was funded by the Academy of Finland, grant numbers 138329, 134684 and 258978.

1. Cross-linking of Strep-Tactin Polymethacrylate Resin with Bis(sulfosuccinimidyl) Suberate (BS3)
<ol>
	<li>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.</li>
	<li>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 &#181;l of the suspension to a spin column using a pipette tip with the...

<ol>
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Vis. Exp. (55), (2011).</li><li>Witte, C. P., Noel, L. D., Gielbert, J., Parker, J. E., Romeis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+one-step+protein+purification+from+plant+material+using+the+eight-amino+acid+StrepII+epitope.">Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope.</a> Plant Mol. Biol. 55 (1), 135-147 (2004).</li><li>Werner, A. K., Sparkes, I. A., Romeis, T., Witte, C. 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No conflicts of interest declared.

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...

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&#160;&#176;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).

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr>
			<td>Bis(sulfosuccinimidyl) suberate (BS3), No-Weigh format, 8 x 2mg</td>
			<td>Pierce/Thermo Scientific</td>
			<td>21585</td>
			<td><a href="http://www.fishersci.com">www.fishersci.com CAUTION: Hazardous substance. Causes serious respiratory, skin and eye irritation. Wear protective gloves and eye protection.</a></td>
		</tr>
		<tr>
			<td>Strep-Tactin MacroPrep resin (50% suspension)</td>
			<td>IBA</td>
			<td>2-1505</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter, cellulose acetate membrane, pore size 0.45 &#956;m, non-sterile</td>
			<td>Costar (Corning)</td>
			<td>8163</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Dolphin-nose tubes</td>
			<td>Costar (Corning)</td>
			<td>3213</td>
			<td><a href="http://www.corning.com/lifesciences/">www.corning.com/lifesciences/</a></td>
		</tr>
		<tr>
			<td>Avidin&#160;</td>
			<td>IBA</td>
			<td>2-0204</td>
			<td><a href="http://www.iba-lifesciences.com/">www.iba-lifesciences.com</a></td>
		</tr>
	</tbody>
</table>

one-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (bs3)

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.

The purification procedure is schematically illustrated in Figure 1, together with a representation of problems associated with other existing purification methods.
<img alt="Figure 1" fo:content-width="5.5in" src="/files/ftp_upload/51536/51536fig1highres.jpg" width="650" /> 
Figure 1. Schematic representation of the purification procedure. The workflow includes stabilization of the resin-coupled Strep-Tactin tetramer...

Watch this Scientific Journal Video about One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bissulfosuccinimidyl Suberate BS3 at JoVE.com

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

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.

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

Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for ...

one-step-purification-twin-strep-tagged-proteins-their-complexes-on

Research

JoVE Journal

Biology

18.5K 조회수.  University of Helsinki.  방법은 (연쇄상 구균 - Tactin) 수지가 공유 비스 (술포 숙신 이미 딜) 수베 (BS3)와 가교 수정 스트렙 타비 딘에 트윈 패 혈성 태그가 융합 단백질 및 특정 단지의 효율적인 정제에 설명되어 있습니다. 방법은 빠른 속도, 좋은 표적 단백질의 회수 및 고순도의 이점을 갖고, 질량 분석법에 의한 후속 분석과 호환된다. 