This project was funded by the Knut and Alice Wallenberg Foundation and the Swedish Research Council.

1. Cloning of the target gene/ABDz1-tagged fusion construct
<ol>
 <li>Prepare PCR fragments of the ABDz1 gene flanked by suitable restriction sites for N-terminal ligation of the target gene in the expression plasmid (a plasmid containing ABDz1 is freely available through a material transfer agreement).</li>
 <li>Cleave purified expression vector and PCR fragments with chosen restriction enzymes in a suitable reaction buffer. Purify the products before ligation.</li>
 <li>Ligate the restricted ABDz1 fragment into the expression vector containing the gene of interest and transform the ligation product to E. coli (in the original method the RR1&Delta;M15 strain was used8). Spread transformed cells on agar plates supplemented with suitable antibiotics for selection.</li>
 <li>PCR screen a few colonies and sequence verify the resulting expression cassette by DNA sequencing.</li>
 <li>Prepare plasmid from an overnight culture of a sequence verified colony and transform to the preferred expression strain (in the original method E. coli Rosetta (DE3), hosting a pRARE plasmid to improve the production of proteins encoded by human genes, were used).</li>
</ol>
2. Protein expression
<ol>
 <li>Inoculate a single bacterial colony into 10 ml of tryptic soy broth supplemented with 5 g/l yeast extract and appropriate antibiotics. Incubate at 150 rpm at 37 &deg;C overnight.</li>
 <li>Inoculate 1 ml of overnight culture in 100 ml fresh medium and induce protein expression (originally 1 mM isopropyl-&beta;-D-thiogalactoside when a lac operon was utilized) when the cells reach the logarithmic growth phase.</li>
 <li>Incubate at 150 rpm at 25 &deg;C overnight and harvest cells by centrifugation.</li>
</ol>
3. Orthogonal affinity purification
<ol>
 <li>Resuspend the pellet in 25 ml running buffer (25 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0.05 % (w/v) Tween 20, pH 8.0) and disrupt the cells by sonication at 60 % amplitude and 1.0/1.0 pulses for 3 min. Centrifuge the sample and filter the target protein containing supernatant (0.45 &mu;m) before further purification.</li>
 <li>Equilibrate either a 1 ml HSA Sepharose column or an NHS-activated column, previously coupled with HSA according to the supplier's recommendations, with 10 column volumes (CV) of running buffer at 1 ml/min on a suitable protein purification system. Load the bacterial lysate at 0.5 ml/min and then reset the flow rate to 1 ml/min.</li>
 <li>Wash the column with 5 CV of running buffer followed by 5 CV of washing buffer (5 mM NH4Ac, pH 5.5).</li>
 <li>Elute the sample with elution buffer (0.5 M HAc, pH 2.5) at 1 ml/min and collect fractions, monitor absorption at 280 nm to select fractions from the eluted peak for further purification.</li>
 <li>Pool the fractions with the highest protein concentration, dilute two times in SuRe running buffer (20 mM phosphate, 150 mM NaCl, pH 7.2) and make sure the pH is around neutral. Add 1 M Tris-HCl pH 8 to increase pH if necessary.</li>
 <li>Load the sample at 0.5 ml/min on a 1 ml HiTrap MabSelect SuRe column that has been equilibrated with 10 CV of SuRe running buffer at 1 ml/min. Reset the flow rate to 1 ml/min after loading.</li>
 <li>Wash the column with 5 CV of SuRe running buffer (step 5) and elute the proteins with 0.2 M HAc, pH 2.7. For sensitive target proteins the eluate can be neutralized directly upon collection by addition of Tris-HCl.</li>
</ol>
If the chromatographic steps are reversed the eluate from the MabSelect SuRe column can be diluted in running buffer (step 3.1) and the pH increased to around 8 by addition of 1 M Tris-HCl. In general, narrower peaks are observed when the SuRe matrix is employed in the second step.
4. Evaluation of purity by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
<ol>
 <li>Load the purified fractions on a reducing SDS-PAGE and, if desired, analyze the molecular weight of the purified product by mass spectrometry. It is important to fully reduce the sample since a free cysteine in ABDz1 may cause dimerization of the product under non-reducing conditions.</li>
</ol>
5. Representative Results:
As a proof of principle, orthogonal affinity purification facilitated by the ABDz1 tag was evaluated for three human target proteins representing different solubility classes, molecular weights and isoelectric points (Table 1). The ABDz1 gene was genetically fused to the target genes and the constructs were expressed in E. coli, Figure 2 shows a flow chart for all steps in the method consecutively. Following purification by the orthogonal protocol, samples collected at different points were analyzed by SDS-PAGE (Figure 3). The results clearly show the utility of a dual tag and two highly specific purification steps. Even though reasonable purity is achieved after the initial purification step as seen from the gels, the successive step yields a very pure product well suited for highly demanding applications.
<img alt="Figure 1" src="/files/ftp_upload/3370/3370fig1.jpg" /> 
Figure 1. Design of the combinatorial library used for selection of the bispecific ABDz1 tag. 
The 46 amino acid albumin-binding domain folds into a stable three helix bundle and it contains a binding site for human serum albumin mainly located to the second helix. By genetically randomizing eleven surface exposed amino acids located in the first- and third helix, a novel binding surface was engineered. The eleven positions are indicated in the figure and numbered according to Kraulis et al.9 . Following biopanning against a dimer of the Z-domain of Staphylococcal protein A with the combinatorial library expressed on phage, the bispecific ABDz1 molecule was identified.
<img alt="Figure 2" src="/files/ftp_upload/3370/3370fig2.jpg" /> 
Figure 2. A simplified flow chart for the orthogonal affinity purification protocol. 
A PCR-fragment containing the ABDz1 sequence is ligated (N-terminally) with a gene of interest in a suitable expression vector. The ligation product is transformed to E. coli for sequence verification and plasmid preparation. Following transformation to an expression host, a large-scale culture for recombinant protein expression is set up from an initial overnight culture. Bacteria are harvested by centrifugation and lysed by sonication to produce bacterial lysate containing the target protein. After a filtration step to remove residual solid particles, the lysate is subjected to orthogonal affinity purification on a liquid handling system by undergoing two purification steps in succession. Eluted peaks from both purification steps are sampled and evaluated by SDS-PAGE to assess purity and compared to the lysate before purification.
<img alt="Figure 3" src="/files/ftp_upload/3370/3370fig3.jpg" /> 
Figure 3. SDS-PAGE analysis of target proteins expressed and purified in fusion with ABDz1. 
Samples taken from the bacterial lysate of three proteins expressed in fusion to ABDz1, representing different properties, and the ABDz1 tag itself were collected together with samples from the corresponding peaks from purification on a HSA-column followed by a MabSelect SuRe-column (A). Lanes 1-4 represent samples from lysates before purification, lanes 5-8 from the HSA-purification (first step) and lanes 9-12 from the MabSelect SuRe purification (second step). Samples are loaded in the following order: ABDz1-141377, ABDz1-HT875, ABDz1-HT2375 and ABDz1. In addition, samples acquired from the same lysates purified in the reverse order on the same columns were analyzed (B). Lanes 1-3 represent samples from lysates before purification, lanes 4-6 from MabSelect SuRe-purification (first step) and lanes 7-9 from HSA-purification (second step). Samples were loaded in the same order as in (A) but the tag itself is not included. From those results it is clear that this two-step method yields highly purified proteins regardless of the order in which the steps are applied.
<table border="1">
 <tbody>
 <tr>
 <td>Nameb</td>
 <td>Target protein Uniprotc</td>
 <td>Molecular weight (kDa)</td>
 <td>Solubility classd</td>
 <td>Molecular weight of fusion product (kDa)</td>
 <td>Isoelectric point of fusion product</td>
 </tr>
 <tr>
 <td>141377</td>
 <td>B7Z315</td>
 <td>17.4</td>
 <td>4</td>
 <td>23.7</td>
 <td>8.5</td>
 </tr>
 <tr>
 <td>HT875</td>
 <td>P01040</td>
 <td>10.8</td>
 <td>3</td>
 <td>17.1</td>
 <td>4.9</td>
 </tr>
 <tr>
 <td>HT2375</td>
 <td>P00740</td>
 <td>8.9</td>
 <td>5</td>
 <td>15.2</td>
 <td>6.8</td>
 </tr>
 </tbody>
</table>
<ol class="lalpha">
 <li>Molecular weight of ABDz1 tag alone is 6.3 kDa, isoelectric point 6.7.</li>
 <li>Target protein represents a part of the Uniprot protein.</li>
 <li><a href="http://www.uniprot.org">http://www.uniprot.org</a>.</li>
 <li>Solubility class of protein fragment with N-terminal His6ABP10 .</li>
</ol>
Table 1. Target proteins and fusion products with ABDz1 taga evaluated in a proof of principle study.
Three unique human proteins with different molecular weight, solubility and isoelectric point were chosen for expression and purification by the orthogonal affinity approach.

<ol>
	<li>Waugh, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+the+most+of+affinity+tags.">Making the most of affinity tags.</a> Trends Biotechnol. 23, 316-320 (2005).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+engineering+strategies+for+selective+protein+purification.">Protein engineering strategies for selective protein purification.</a> CET. 28, 1315-1325 (2005).</li><li>Porath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+chelate+affinity+chromatography,+a+new+approach+to+protein+fractionation.">Metal chelate affinity chromatography, a new approach to protein fractionation.</a> Nature. 258, 206-213 (1975).</li><li>Linhult, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+the+interaction+between+albumin-binding+domain+from+streptococcal+protein+G+and+human+serum+albumin.">Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin.</a> Protein Sci. 11, 206-213 (2002).</li><li>Nilsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+IgG-binding+domain+based+on+staphylococcal+protein.">A synthetic IgG-binding domain based on staphylococcal protein.</a> A. Protein Eng. 1, 107-113 (1987).</li><li>Alm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+bispecific+protein+selected+for+orthogonal+affinity+purification.">A small bispecific protein selected for orthogonal affinity purification.</a> Biotechnol. J. 5, 605-617 (2010).</li><li>Nilsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+affinity+domains+for+the+detection,+purification+and+immobilization+of+recombinant+proteins.">Multiple affinity domains for the detection, purification and immobilization of recombinant proteins.</a> J. Mol. Recognit. 9, 585-594 (1996).</li><li>R&#252;ther, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pUR+250+allows+rapid+chemical+sequencing+of+both+DNA+strands+of+its+inserts.">pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts.</a> Nucleic Acids Res. 10, 5765-5772 (1982).</li><li>Kraulis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serum+albumin-binding+domain+of+streptococcal+protein+G+is+a+three-helical+bundle:+a+heteronuclear+NMR+study.">The serum albumin-binding domain of streptococcal protein G is a three-helical bundle: a heteronuclear NMR study.</a> FEBS Lett. 378, 190-194 (1996).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+flow+cytometry-based+method+for+analysis+of+expression+levels+in+E+coli+giving+information+about+precipitated+and+soluble+protein.">Novel flow cytometry-based method for analysis of expression levels in E coli giving information about precipitated and soluble protein.</a> J. Biotechnol. 119, 133-146 (2005).</li><li>Carter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+proteolysis+of+fusion+proteins.">Site-specific proteolysis of fusion proteins.</a> ACS Sympos. Ser. 427, 181-193 (1990).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negatively+charged+purification+tags+for+selective+anion-exchange+recovery.">Negatively charged purification tags for selective anion-exchange recovery.</a> PEDS. 17, 779-786 (2004).</li><li>Gr&#228;slund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+engineering+of+a+protein+domain+to+allow+efficient+ion-exchange+recovery.">Charge engineering of a protein domain to allow efficient ion-exchange recovery.</a> Protein Eng. 13, 703-709 (2000).</li></ol>

No conflicts of interest declared.

The orthogonal affinity purification protocol presented here enables efficient purification of a wide range of target proteins. By combining the inherent binding site of the albumin-binding domain with a novel binding surface, a small bispecific protein tag was developed. It is very straightforward to utilize the tag since it can simply be cloned and expressed in fusion to any protein of interest in any preferred expression vector. Standard equipment available in most laboratories can be employed for the two-step protein purification. Since the function of the ABDz1 tag depends on correct folding of the domain to efficiently expose its two binding surfaces, the method is limited to proteins expressed in soluble form and not suited for purifications under denaturing conditions. Reducing agents should also be avoided since they interfere with the binding of ABDz1 to the Z-domain on the MabSelect SuRe matrix.
Orthogonal affinity purification provides a useful alternative to traditional methods in order to satisfy the requirements of highly purified proteins in many demanding applications. At the same time, this method eliminates the need for combinatorial tagging when different purification strategies are used in succession. For applications requiring a native target protein, a protease cleavage site may be introduced to render enzymatic removal of the ABDz1 tag possible11 . Furthermore, the ABDz1 tag is the first bispecific small and folded protein domain described to date. It is unique since it provides a purification strategy that depends on two target specific affinity interactions. In the future it would be interesting to expand this concept by developing novel bispecific tags that carry new binding surfaces that are compatible with readily available and inexpensive chromatographic resins. For example, by replacing the amino acids involved in albumin binding with basic residues, a tag compatible with ion exchange chromatography may be achievable. A similar concept has been proven effective by charge engineering of the Z-domain to generate tags known as Zacid12 and Zbasic13 compatible with anion- and cation exchange, respectively.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>E. coli RR1&Delta;M15</td>
			<td>American Type Culture Collection</td>
			<td>35102</td>
		</tr>
		<tr>
			<td>E. coli Rosetta (DE3)</td>
			<td>Novagen</td>
			<td>70954</td>
		</tr>
		<tr>
			<td>Tryptic soy broth</td>
			<td>Difco</td>
			<td>211822</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Difco</td>
			<td>212720</td>
		</tr>
		<tr>
			<td>Vibra cell sonicator</td>
			<td>Sonics and materials</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HSA Sepharose</td>
			<td>Pharmacia Biotech</td>
			<td>Former product</td>
		</tr>
		<tr>
			<td>HiTrap MabSelect SuRe</td>
			<td>GE Healthcare</td>
			<td>11-0034-93</td>
		</tr>
		<tr>
			<td>HiTrap NHS-activated HP column</td>
			<td>GE Healthcare</td>
			<td>17-0716-01</td>
		</tr>
	</tbody>
</table>

orthogonal protein purification facilitated by a small bispecific affinity tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

Watch this Scientific Journal Video about Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag at JoVE.com

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

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17.0K Views.  Royal Institute of Technology. Un romanzo e altamente efficiente in due fasi cromatografia di affinità protocollo è stato sviluppato ed è descritto in dettaglio. Il metodo si basa su un tag purificazione con due affinità intrinseca ed è applicabile ad una vasta gamma di proteine ​​bersaglio con proprietà diverse.

Video: Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...