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/pubmed?term=((Comparison+of+affinity+tags+for+protein+purification%5BTitle%5D)+AND+%22Protein+Expr.+Purif%22%5BJournal%5D)">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/pubmed?term=((A+method+for+the+separation+of+GST+fusion+proteins+from+co-purifying+GroEL%5BTitle%5D)+AND+%22Trends+Genet%22%5BJournal%5D)">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/pubmed?term=((Expression+of+a+recombinant+form+of+the+V+antigen+of+Yersinia+pestis%2C+using+three+different+expression+systems%5BTitle%5D)+AND+%22Vaccine%22%5BJournal%5D)">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/pubmed?term=((Selective+adsorption+of+poly-His+tagged+glutaryl+acylase+on+tailor-made+metal+chelate+supports%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">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/pubmed?term=((The+baculovirus+expression+vector+pBSV-8His+directs+secretion+of+histidine-tagged+proteins%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">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/pubmed?term=((Cooperation+of+breast+cancer+proteins+PALB2+and+piccolo+BRCA2+in+stimulating+homologous+recombination%5BTitle%5D)+AND+%22Nat.+Struct.+Mol.+Biol%22%5BJournal%5D)">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/pubmed?term=((Isoforms+of+Serratia+marcescens+nuclease.+The+role+of+Mg2%2B+in+the+hydrolysis+mechanism%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">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/pubmed?term=((The+breast+cancer+tumor+suppressor+BRCA2+promotes+the+specific+targeting+of+RAD51+to+single-stranded+DNA%5BTitle%5D)+AND+%22Nat.+Struct.+Mol.+Biol%22%5BJournal%5D)">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/pubmed?term=((Interactions+between+BRCA2+and+RAD51+for+promoting+homologous+recombination+in+Leishmania+infantum%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">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/pubmed?term=((Baculovirus+expression+vector+system%3A+an+emerging+host+for+high-throughput+eukaryotic+protein+expression%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">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 th...

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><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;E.Coli &lt;/em&gt;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&#039;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 colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</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 Pr...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

42.3K 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

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

Biochemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...

Strategies for Expressing and Purifying Solute Carrier Transporters

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

Optimizing FPLC for Efficient Purification of His-Tagged FEN1 Protein

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

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 ...

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 ...

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 a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

Immunology and Infection

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

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

Summary

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