We thank Anne-Marie Dion-Cote for discussions leading to the development of the method. J.K. and R.B. are FQNRT doctoral scholars, M.-M.G. is a Vanier CIHR scholar, and J.-Y.M. is a FRSQ senior researcher. This work was supported by funds from the Natural Sciences and Engineering Research Council to J-Y. M.

1. Production of Recombinant Baculovirus
Baculoviruses are generated using the Bac-to-Bac Baculovirus Expression System of Invitrogen mainly in accordance with the manufacturer&#39;s protocol with only slight modifications:
<ol>
	<li>A modified pFastBac1 vector (Gibco, life) was created containing the GST and His-tags (Figure 2). The MultiCloning Site (MCS) of a pET-52b(+) vector (Novagen), containing a 10xHis-tag, was inserted into the MCS of a pFastBac1 vector to generate pFastBac1-Strep-10xHis. The pET-52b(+) MCS sequence was PCR amplified between the XbaI and BlpI restriction sites, adding a BglII restriction site at the 5&#39; extremity and a HindIII restriction site at the 3&#39; extremity. The PCR product was then cloned between BamHI and HindIII restriction site of pFastBac1, using the compatibility of BamHI and BglII restriction sequences. Since the aim was to generate a pFastBac1 allowing the expression of recombinant protein with streptavidin- and 10xHis-tags, the BamHI restriction site of the pET-52b(+) MCS sequence was not used. Also, the XbaI restriction site was not used to avoid the redundancy of restriction sites occurring between the sites already in the MCS of pFastBac1 and these inserted with the MCS of pET-52b(+). The GST coding sequence with the PreScission Protease recognition site (LeuGluValLeuPheGln/GlyPro) was then inserted into pFastBac1-Strep-10xHis. The GST cDNA sequence from pGEX-6P-2 (GE Healthcare) was PCR amplified with primers containing NcoI and KpnI restriction sites at the 5&#39; and 3&#39; extremities, respectively. The NcoI site allows the addition of a start codon but also an Alanine to the GST. The PCR product was then cloned into pFastBac1-Strep-10xHis between the NcoI and KpnI restriction sites, resulting in the deletion of the Streptavidin-tag. The new fusion vector thus obtained was named pFastBac1-GST-10xHis.</li>
	<li>Clone the cDNA coding for the protein of interest into the pFastBac-GST-10xHis vector between the GST (N-terminal) and the His-tag (C-terminal). Be careful to remove the cDNA stop codon to allow the fusion with the C-terminal His-tag.</li>
	<li>Transform E. coli DH10Bac with the recombinant pFastBac (obtained in step 1.2) to generate the bacmid. Allow colonies to grow overnight at 37 &#176;C and several hours at 30 &#176;C on selection plates containing Bluo-gal and IPTG (10 &#956;g/ml tetracyclin, 50 &#956;g/ml kanamycin, 7 &#956;g/ml gentamycin, 100 &#956;g/ml Bluo-Gal, 40 &#956;g/ml IPTG).</li>
	<li>Pick and restreak 2 white colonies (from step 1.3) on selection plates to confirm the white phenotype.</li>
	<li>Inoculate 2 ml of LB containing 10 &#956;g/ml tetracyclin, 50 &#956;g/ml kanamycin, 10 &#956;g/ml gentamycin with the two white colonies from step 1.4 and let them grow overnight under agitation (250 rpm) at 37 &#176;C.</li>
	<li>In order to purify the bacmid DNA, pellet the bacteria from step 1.5, resuspend the cells in 300 &#956;l of solution I and add 300 &#956;l of solution II (Maxiprep kit from Qiagen). Incubate for 5 min at room temperature, add 300 &#956;l of 3 M KOAc pH 5.5 and incubate for 10 min on ice. Centrifuge the samples at 4 &#176;C, maximum speed for 10 min. Add 800 &#956;l of isopropanol to the supernatants and incubate for 10 min on ice. Centrifuge the samples for 15 min at 4 &#176;C and wash the pellet with 500 &#956;l of 70% ethanol. Let the pellet dry and resuspend it under sterile conditions in 40 &#956;l of Tris-EDTA pH 8.0. The bacmid DNA should be stored at 4 &#176;C.</li>
	<li>Generate baculoviruses as described in the manufacturer&#39;s protocol. Baculoviruses can be stored at 4 &#176;C protected from light.</li>
</ol>
2. Recombinant Protein Expression
<ol>
	<li>In order to choose the most efficient baculovirus for purification and to monitor what time the peak of protein expression is reached, perform a mini-infection assay as follows: Infect 2 x 107 of Sf9 infected cells cultured at 27 &#176;C in Grace&#39;s media (complemented with 10% FBS and 1% P/S) with 133 &#956;l (1/150) of baculovirus. Collect aliquots of 1.5 ml at 0, 24, 48, and 72 hr post-infection. Pellet the cells and analyze the expression level of the protein of interest by western blotting using anti-tag antibodies.</li>
	<li>Use the most efficient baculovirus from step 2.1 to infect a spinner containing 1.0 x 106 Sf9 cells per ml in 500 ml of media with a ratio of 1/150 between virus and media. Let the infected cells grow in suspension at 27 &#176;C and harvest the cells when the maximum protein expression (as determined in step 2.1 above) is reached. Pelleted cells can be stored at -80 &#176;C until further usage.</li>
</ol>
3. Preparation of Soluble Cell Lysate
All of the following incubation steps are carried out at 4 &#176;C under mild rotation.
<ol>
	<li>Lyse Sf9 cells expressing your protein of interest in ~20 ml of GST binding buffer (150 mM NaCl, 1 mM EDTA, 0.05% Triton-X-100, 1 mM DTT in PBS1X for a final concentration of NaCl of 250 mM, and protease inhibitor cocktail (Roche). In an ice water bath, dounce the solution 20x with a dounce homogenizer, sonicate 3x 30 seconds each (70% output), and dounce again 20x.</li>
	<li>(Optional). Incubate the total cell lysate with 1 mM MgCl2 and benzonase7 nuclease (2.5 U/ml) for 30 min at 4 &#176;C to remove DNA or RNA contamination.</li>
	<li>Centrifuge at 18,000 rpm for 30 min at 4 &#176;C. Keep the supernatant.</li>
	<li>(Optional). Centrifuge the supernatant again for 30 min at 18,000 rpm at 4 &#176;C to get a clear soluble lysate. Pass the supernatant through a 0.2 or 0.45 &#956;m&#160;filter to avoid clogging.</li>
</ol>
4. Binding of Protein on GST Beads
<ol>
	<li>Incubate the soluble cell lysate with 1 ml of GST beads (prewashed two times with 10 ml of GST binding buffer) for 1 hr at 4 &#176;C under gentle rotation.</li>
</ol>
Comment: The incubation time needs to be optimized according to the stability of the protein and binding efficiency.
<ol start="2">
	<li>Quick spin at 700 rpm and remove supernatant containing unbound proteins (this can be kept for further analysis). Wash the GST-bound proteins (Figure 3B, lane 1) with GST washing buffer (GST binding buffer with 350 mM NaCl final).</li>
	<li>Repeat step 4.2 2x. At the last wash, remove as much supernatant as possible.</li>
	<li>Incubate the beads with 5 mM ATP and 15 mM MgCl2 (in 10 ml GST binding buffer) for 1 hr at 4 &#176;C to avoid nonspecific binding of heat-shock proteins8. Wash the beads three times with GST washing buffer.</li>
</ol>
5. PreScission Cleavage of the GST
<ol>
	<li>Centrifuge the GST beads, remove the supernatant and wash the GST beads with P5 buffer (50 mM NaHPO4 pH 7.0, 500 mM NaCl, 10% glycerol, 0.05% Triton-X-100, 5 mM imidazole).</li>
	<li>Incubate the GST-beads with PreScission enzyme in P5 buffer from 3 hr to overnight at 4 &#176;C. (Divide the GST beads in fractions of 100 &#956;l and add 4-8 units (2-4 &#956;l) of enzyme diluted in 100 &#956;l of P5 buffer).</li>
</ol>
Comment: The incubation time of the PreScission step needs to be optimized according to the molecular weight as well as the stability of the protein. If degradation is observed, this incubation time can be decreased to a minimum of 2 hr instead of overnight.
<ol start="3">
	<li>Quick spin and collect the supernatant. Repeat this step two times after adding 100 &#956;l of P5 buffer to the beads and pool all fractions (Figure 3B, lane 2).</li>
</ol>
Comment: The remaining beads can be used for analysis of cleavage efficiency (Figure 3B, lane 3). Usually, 70-80% of the protein is cleaved.
6. Protein-binding to TALON Metal Affinity Resin
<ol>
	<li>Divide the elution from above into 3 fractions of 1 ml and incubate each with 100 &#956;l&#160;Talon metal affinity resin dry beads (prewashed two times with P5 buffer) for 1 hr under rotation.</li>
</ol>
Comment: Dividing the elution in several fractions increases binding/washes efficiency.
<ol start="2">
	<li>Wash the resin 5 min with P30 buffer (P5 buffer with a final concentration of 30 mM imidazole).</li>
	<li>Repeat step 6.2 2x and pool the beads in a single tube. Before the last wash, remove as much supernatant as possible.</li>
</ol>
Comment: This corresponds to the TALON bound sample (Figure 3B, lane 4).
7. Elution of the Purified Protein
<ol>
	<li>Incubate the protein bound TALON resin for 5 min under rotation with P500 buffer (P5 buffer with a final concentration of 500 mM imidazole). Use a ratio between buffer and beads of 1:1 v/v. (For example, if you had taken 200 &#956;l&#160;of dry beads, you will have to add 200 &#956;l&#160;of P500). Repeat this step two times and keep each eluted fraction separately (named elution 1 (E1), elution 2 (E2) and elution 3 (E3); Figure 3B, lanes 5-7).</li>
</ol>
Comment: The remaining beads can be used for analysis of elution efficiency (Figure 3B, lane 8). Typically, 60-80% of the protein is eluted in total.
<ol start="2">
	<li>Analyze the quantity and quality of your purified protein by loading approximately 20-40 &#956;l&#160;of each fraction with a standard BSA concentration on a SDS-PAGE and stain with Coomassie blue (Figure 3B) or SYPRO protein stain for visualization.</li>
</ol>
Comment: The protein of interest can also be detected throughout the purification procedure using anti-GST and anti-His antibodies (Figure 3C).
8. Storage of the Purified Protein
<ol>
	<li>Dialyze the purified protein against an appropriate storage buffer (e.g. 20 mM Tris Acetate pH 8.0, 200 mM KAc, 10% Glycerol, 1 mM EDTA, 0.5 mM DTT) at 4 &#176;C. It is important to check for precipitation of the purified protein during the dialysis. We usually dialyze 2x for 1 hr at 4 &#176;C under agitation rotation.</li>
	<li>Make small aliquots of the purified protein (10-20 &#956;l) and freeze on dry ice for 30 min. Store the aliquots at -80 &#176;C and avoid many thaw/freezing cycles.</li>
</ol>

<ol>
	<li>Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J., Tan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+affinity+tags+for+protein+purification.">Comparison of affinity tags for protein purification.</a> Protein Expr. Purif. 41, 98-105 (2005).</li><li>Thain, A., Gaston, K., Jenkins, O., Clarke, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+separation+of+GST+fusion+proteins+from+co-purifying+GroEL.">A method for the separation of GST fusion proteins from co-purifying GroEL.</a> Trends Genet. 12, 209-210 (1996).</li><li>Carr, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+recombinant+form+of+the+V+antigen+of+Yersinia+pestis,+using+three+different+expression+systems.">Expression of a recombinant form of the V antigen of Yersinia pestis, using three different expression systems.</a> Vaccine. 18, 153-159 (1999).</li><li>Armisen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+adsorption+of+poly-His+tagged+glutaryl+acylase+on+tailor-made+metal+chelate+supports.">Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports.</a> J. Chromatogr. A. 848, 61-70 (1999).</li><li>Kuhn, S., Zipfel, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+baculovirus+expression+vector+pBSV-8His+directs+secretion+of+histidine-tagged+proteins.">The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins.</a> Gene. 162, 225-229 (1995).</li><li>Buisson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+of+breast+cancer+proteins+PALB2+and+piccolo+BRCA2+in+stimulating+homologous+recombination.">Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.</a> Nat. Struct. Mol. Biol. 17, 1247-1254 (2010).</li><li>Filimonova, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoforms+of+Serratia+marcescens+nuclease.+The+role+of+Mg2++in+the+hydrolysis+mechanism.">Isoforms of Serratia marcescens nuclease. The role of Mg2+ in the hydrolysis mechanism.</a> Biochemistry. 62, 983-988 (1997).</li><li>Thorslund, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+breast+cancer+tumor+suppressor+BRCA2+promotes+the+specific+targeting+of+RAD51+to+single-stranded+DNA.">The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA.</a> Nat. Struct. Mol. Biol. 17, 1263-1265 (2010).</li><li>Genois, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+BRCA2+and+RAD51+for+promoting+homologous+recombination+in+Leishmania+infantum.">Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.</a> Nucleic Acids Res. 40, 6570-6584 (2012).</li><li>Shrestha, B., Smee, C., Gileadi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+expression+vector+system:+an+emerging+host+for+high-throughput+eukaryotic+protein+expression.">Baculovirus expression vector system: an emerging host for high-throughput eukaryotic protein expression.</a> Methods Mol. Biol. 439, 269-289 (2008).</li></ol>

The GST-His purification protocol presented here is suitable for the purification of a broad range of sizes of recombinant proteins: We successfully purified LiRAD51 (41kDa), piBRCA2 (120kDa), PALB2 (130kDa), and an unstable high molecular weight protein of Leishmania infantum: LiBRCA2 (125kDa)6,9. Moreover, the proteins purified with this protocol were biochemically active6. The success of this method solely depends on the expression, solubility and stability of the desired protein.
	Molecular biology vectors for expression in other hosts, such as E. coli or yeast, can be easily modified using the widely available GST and His tags. This highlights the applicability and cost-effectiveness of the present technique for most molecular biology labs. A variety of advantages influenced our decision to choose infected cells over bacterial or mammalian cells for recombinant protein expression and purification. For example, posttranslational modifications such as methylation, phosphorylation and ubiquitinylation can strongly influence the enzymatic function of a protein10. Hence, to study the physiological properties of a protein, it is favorable to use a system that allows for posttranslational modification, such as Sf9 infected cells. Another advantage of using Sf9 over mammalian cells, for instance, is of economical nature, as the infected cell system requires considerably fewer cells and no costly transfection method compared to the mammalian system.
	The simplicity of the presented method will help researchers to obtain a protein or protein complex in a highly purified form with standard lab equipment, which is advantageous for a lab with minimal equipment.

The purification of recombinant proteins is crucial to address fundamental questions in biochemistry. Conventional ways of protein purification like ion exchange chromatography and size exclusion chromatography rely on physical properties of the target protein such as its isoelectric point and charge or size, respectively. The latter protein characteristics are shared by a variety of proteins, which increases considerably the chance of contaminating proteins in conventional protein purification strategies. This problem may be circumvented with the use of multiple purification columns, which is time consuming. At the same time, the latter chromatography methods demand expensive experimental setup. Affinity-tag purification strongly increases target-specificity, as in most of the cases the tag will be unique to the protein of interest. In recent studies, Flag- or HA-affinity purification has been widely used.
In contrast to existing recombinant protein purification protocols in which single tags are used, we established the unique combination of two tags. Our method involves the fusion of a GST-tag at the N-terminus and a His-tag at the C-terminus of the protein of interest, for an optimal ratio between quantity and purity of the desired protein. The GST is a long tag (29 kDa), which is highly efficient for purification on glutathione Sepharose beads. Furthermore, using GST guarantees cost-effectiveness of our method1. The possibility to cleave off the GST with the PreScission enzyme (with recognition sequence LeuGluValLeuPheGln/GlyPro, resulting in the addition of only two amino acids) has many advantages, for instance, this strategy avoids alterations of the physiological protein functions due to allosteric hindrance. The small His-tag fused to the other protein extremity serves in a second purification step to increase protein purity by washing off the cleaved GST, as well as degraded proteins and other contaminants. Additionally, the protocol does not require a dialysis step when switching from the GST to the His-purification step column (TALON resin). Common contaminants in such purification processes are Heat Shock Proteins (HSP). The addition of an incubation step with MgCl2 and ATP allows the removal of these contaminants (Figure 1).
Fast Protein Liquid Chromatography (FPLC) and High Performance Liquid Chromatography (HPLC) are common techniques that depend on expensive instrumentation to generate high yield of the purified protein of interest. The batch purification protocol we present here, in contrast, is manual and does not require an expensive instrumental setup. A high yield of protein can be reached by scaling up the protocol. At the same time, with the batch purification protocol, the elution volume can be adjusted in order to enhance protein concentration, which is not given with FPLC or HPLC.
Also, with the batch GST-His purification protocol, proteins with a size-range of ~10-300 kDa can be purified. One of the biggest advantages is given by the fact that one can purify several proteins at the same time, for instance, both a wild-type and its mutant protein. The success of the presented protocol solely depends on the expression level and solubility of the protein of interest.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>E.Coli DH10Bac (competent)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bac-to-Bac Baculovirus Expression System</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Maxi Prep Kit</td>
			<td>Qiagen</td>
			<td>12163</td>
			<td>Solution I and II</td>
		</tr>
		<tr>
			<td>Ultra Pure Bluo-Gal</td>
			<td>Gibco (Life)</td>
			<td>15519028</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Gibco (Life)</td>
			<td>15529019</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf9 infected cells</td>
			<td>ATCC</td>
			<td>CRL-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>PreScission enzyme</td>
			<td>GE Healthcare</td>
			<td>27084301</td>
			<td></td>
		</tr>
		<tr>
			<td>Grace&#39;s infected medium supplemented</td>
			<td>Gibco (Life)</td>
			<td>11605-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum characterized</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>P/S (Penicillin-Streptomycin)</td>
			<td>Gibco (Life)</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione Sepharose resin</td>
			<td>GE bioscience</td>
			<td>17075605</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Talon resin</td>
			<td>Clontech</td>
			<td>635504</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>BioShop</td>
			<td>288324</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco (Life)</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal GST-antibody</td>
			<td>Production in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal 6X-His-antibody</td>
			<td>Clontech</td>
			<td>631212</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dounce homogenizer (tight)</td>
			<td>Wheaton</td>
			<td>357546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator (Fisher dismembrator)</td>
			<td>Fisher</td>
			<td>Model 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis bag (50 mm)</td>
			<td>Fisher Scientific</td>
			<td>2115217</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 order to illustrate the efficiency of the GST-His purification protocol, we purified Rec14, a S. pombe protein of 32.9 kDa. The Rec14 cDNA was cloned in our modified pFastBac1 vector allowing the additions of GST- and His-tags at the N- and C- termini, respectively (Figure 1A). Recombinant baculovirus were then prepared and used to infect SF9 infected cells for protein expression. The soluble cell lysates were incubated with GST beads and bound-proteins were eluted by cleaving the GST with PreScission protease. The resulting Rec14-His protein was affinity purified on TALON resin and bound-proteins were eluted with TALON buffer containing 500 mM imidazole. Purified Rec14-His was dialyzed in storage buffer and stored at -80 &#176;C (Figure 1B).
The soluble and total cell lysates from SF9 cells infected with the Rec14 recombinant baculovirus, or mock-infected as control, were analyzed by Coomassie blue staining, allowing us to confirm the expression of GST-Rec14-His protein (Figure 3A). During the purification process, many samples were analyzed to follow the efficiency of the method. Analysis of these samples by Coomassie blue staining (Figure 3B) shows that during the first step of purification, GST-Rec14 was correctly bound to GST beads, with an apparent molecular weight of ~60 kDa due to the fusion of Rec14 (32.9 kDa) with GST (29 kDa) (lane 1). After PreScission cleavage of the GST, GST-free Rec14-His migrates around 37 kDa (lane 2) while the cleaved GST can be visualized on the GST beads (lane 3, around 25 kDa). Despite a portion of GST-free Rec14 that still remained bound to GST beads (lane 3), notable enrichment in Rec14-His was achieved (lane 2, with no contaminant visible on Coomassie blue staining). This step could be improved by increasing the incubation time of the protein with PreScission. Analysis of Rec14-His bound to TALON beads (after step 7.1, lane 4) compared to the proteins eluted after GST purification step (lane 2), shows that a large fraction of Rec14-His is bound to TALON beads. After several washes, Rec14-His was eluted and collected. Three elutions had been performed. The analysis revealed a high purity of Rec14-His (no contaminant visible by Coomassie staining, lanes 5 to 7), and the greatest concentration of Rec14-His in the first elution. Comparison of eluted fractions (lanes 5 to 7) to the TALON beads after elution (lane 8) illustrates the efficiency of the elution, since only few Rec14-His can be detected as bound on beads.
Samples used in Figure 3B were subjected to western blot analysis with anti-GST and anti-His antibodies in order to follow Rec14 throughout the procedure (Figure 3C). The anti-GST blot shows that some GST-Rec14 and GST alone, were present in the eluted proteins from the GST purification step (lane 2), and that contaminants were removed by the His-tag affinity purification step. This blot allows us also to monitor that GST had been efficiently removed from Rec14 by the PreScission treatment and the His-tag purification step (lanes 4 to 8). The anti-His blot aims to detect Rec14. We can thus confirm that GST-Rec14-His and Rec14-His were not completely cleaved and removed from the GST beads (lane 11). Moreover, at a high concentration, Rec14 seems to aggregate (lane 13). In summary, the results presented demonstrate the efficiency of the GST-His purification for the purification of S. pombe Rec14.
<img alt="Figure 1" fo:content-width="3.5in" fo:src="/files/ftp_upload/50320/50320fig1highres.jpg" src="/files/ftp_upload/50320/50320fig1.jpg" /> 
Figure 1. Schematic outline of the GST-His two-step affinity purification method. A. GST-Rec14-His construct cloned into pFastBac (Baculovirus expression vector). B. Two-step affinity purification of GST-Rec14-His from Sf9 soluble cell lysate: GST-binding, followed by PreScission cleavage of the GST, TALON-binding and elution with imidazole. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50320/50320fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50320/50320fig2highres.jpg" src="/files/ftp_upload/50320/50320fig2.jpg" /> 
Figure 2. Schematic representation of pFastBac1-GST-10xHis. GST (green), PreScission protease site (light blue), the multicloning site (red), and the 10-His tag (dark blue) are shown. In lower case are the nucleotide sequences originating from pET-52b(+). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50320/50320fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50320/50320fig3highres.jpg" src="/files/ftp_upload/50320/50320fig3.jpg" /> 
Figure 3. Exemplary result of GST-His purification of Rec14. A. Coomassie staining of total protein fraction (lane 2 &#38; 3) and soluble cell lysate (lane 4 &#38; 5) from mock-treated Sf9 cells (lane 2 &#38; 4) or infected with GST-Rec14-His baculovirus (lane 3 &#38; 5). Rec14-His and GST possess a molecular weight of approximately 32.9 kDa and 29 kDa, respectively, and the GST-Rec14-His fusion protein has a molecular weight of approximately 61.9 kDa. B. Coomassie staining demonstrating each step of protein purification method (for detailed explanation see PROCEDURE) C. Western blot of the fractions following the purification procedure with anti-GST (lane 1 to 8) and anti-His (lane 9 to 16) antibodies. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50320/50320fig3large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins at JoVE.com

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

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. ...

gst-his-purification-two-step-affinity-purification-protocol-yielding

Nanyang Technological University

Research

JoVE Journal

Biology

41.3K Views.  Hôtel-Dieu de Québec. 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.

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

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 ...

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 ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

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.

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 ...

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 ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

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 ...

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. ...