This work was funded by grants from the National Science Foundation (1929346) and the American Cancer Society (RSG-21-028-01). We would also like to thank members of the Balakrishnan laboratory for helpful discussions.

1. Sample preparation
<ol>
	<li>To purify recombinant FEN1, express the construct (pET-FCH-FEN1) in BL21(DE3) cells as previously described by Ononye et al.8.
	<ol>
		<li>Inoculate LB media (Table 1) with 1% overnight culture and grow the cells at 37 &#176;C until the OD reaches 0.6.</li>
		<li>Induce with 0.4 M isopropyl-beta-D-thiogalactoside (IPTG) and grow the culture for an additional 3 h.</li>
		<li>Harvest the cells by centrifuging the culture at 5,000 &#215; g for 15 min at 4 &#176;C. Store the pellet in a -80&#176;C freezer.</li>
		<li>On the day of the purification, remove the cell pellet from the -80 &#176;C freezer, allow it to thaw on ice, and resuspend the pellet in 100 mL of lysis buffer (Table 1). 
		NOTE: The cell lysate should always be placed on ice during handling to prevent proteolysis. Once thawed, the entire purification process must be completed on the same day. The cell lysate should not be stored in the refrigerator overnight.</li>
	</ol>
	</li>
	<li>Transfer the suspension to a 250 mL beaker and sonicate on ice with 30 s ON and 30 s OFF at 35% amplitude for 15 min. 
	NOTE: Consult the manufacturer&#39;s settings to identify the amplitude.</li>
	<li>Transfer the sonicated sample to a centrifuge tube and spin at 27,000 &#215; g for 30 min at 4 &#176;C to obtain a clear lysate. Place the cell lysate on ice in the 4 &#176;C refrigerator until sample injection.</li>
</ol>
2. Preparing affinity columns
<ol>
	<li>Fit the columns (one for the Ni-NTA resin and one for the cobalt-based resin) to a clamp stand such that the columns are held as straight as possible, avoiding any slanting of the columns. Ensure that the bottom of the columns are plugged in with a stopper and add 1-2 mL of starting buffer to the bottom of each column to prevent air bubbles during column packing.</li>
	<li>Shake the resin bottles gently until the resin is fully resuspended. Open the top of the columns and pour in 10 mL of each slurry in a continuous manner to avoid the inclusion of air bubbles. Pour the resin against a metal spatula or glass rod held at the rim of the column to avoid air bubbles in the column.</li>
</ol>
3. Column packing using a flow adaptor
<ol>
	<li>Unplug the stopper from the column and connect the bottom to one end of the tubing. Connect the other end of the tubing to the UV port of the FPLC system.</li>
	<li>Ensure that the bed support housing has been pulled into the flow adaptor body and slide the flow adaptor body onto the top of the column (Figure 2A). With the cam latch in position 1, lower the flow adaptor taking care not to insert it into the buffer (Figure 2B). Switch the cam latch to position 2 (Figure 2B). Connect the tubing to the system and start the flow of the starting buffer for 1-2 min to rid the tubing of air and prevent air bubbles during packing.</li>
	<li>Switch the latch back to position 1 and lower the flow adaptor into the buffer in one smooth motion, avoiding the inclusion of air bubbles, and switch the latch back to position 2. This allows for some buffer to enter the flow adaptor and removes any remaining bubbles.</li>
	<li>Switch the latch to position 3 and tighten the cam base and locking ring (Figure 2B).</li>
	<li>Start the flow of buffer at 10 mL/min to pack the column. Continue this step for 3 min or until the resin has been well packed.</li>
</ol>
4. Priming the sample loop of the FPLC system
<ol>
	<li>Connect the tubing from the top of the sample loop to Loop E on the FPLC system (Figure 3).</li>
	<li>Connect the tubing from the bottom of the sample loop to Waste 2 on the FPLC system.</li>
	<li>Insert one end of the tubing into the column port of the FPLC system and place the other end in the waste bottle.</li>
	<li>Insert the tubing from pump A in filtered double distilled water (ddH2O).</li>
	<li>Double-click on the pump settings using the software (Figure 4A, labeled 2) and select system pump inject loop (Figure 4C).</li>
	<li>Click on the flow rate settings (Figure 4A, labeled 1) and change the flow rate to 10 mL/min. Enter 0% in the %B box (Figure 4B).</li>
	<li>Start the flow to flush the sample loop with water, which will push the sliding seal downwards.</li>
	<li>Once the sliding seal reaches the bottom of the sample loop, stop the flow. Switch pump A from ddH2O to the starting buffer. Connect the tubing from the top of the sample loop to Waste 2 and that from the bottom to Loop E. Leave the tubing from the column port in the waste.</li>
	<li>Start the flow at 10 mL/min. Once the sliding seal pushed upwards by the incoming starting buffer reaches the top of the sample loop, stop the flow. The sample loop is now ready for sample injection.</li>
</ol>
5. Sample injection
<ol>
	<li>Double-click on the pump settings and select Manual load loop (Figure 4C).</li>
	<li>Unscrew the tubing from Loop E and place it in the waste container.</li>
	<li>Connect the tubing from the column port to the tubing on top of the flow adaptor.</li>
	<li>Draw up the sample using a 30 mL syringe; then, attach the syringe to the injection port adaptor.</li>
	<li>Screw the syringe into the injection port and inject the sample, ensuring that it is visibly injected into the sample loop. Store 100 &#181;L of the sample (label input lysate) in a microcentrifuge tube at -20 &#176;C for SDS-PAGE analysis 
	NOTE: Depending on the total sample volume, 2-3 injections may be required.</li>
	<li>Plug the injection port with the column cap.</li>
	<li>Connect the tubing from the bottom of the sample loop back into the Loop E port.</li>
	<li>Place the tubing from pump B in the elution buffer.</li>
</ol>
6. Method creation and analysis using the FPLC system-linked software
<ol>
	<li>Navigate to the Home tab and select New method.</li>
	<li>Select the Method settings tab on the left column, navigate to the Column type dropdown menu, and select Custom (Figure 5).</li>
	<li>Enter the column volume (5 mL).</li>
	<li>Enter the required flow rate for the run (1 mL/min).</li>
	<li>Navigate to Unit selection and select CV (column volumes) as the method base unit.</li>
	<li>Ensure that Inlet A is set to Buffer A&#160;and Inlet B is set to Buffer B&#160;(the default setting).</li>
	<li>Select the Method Outline tab on the left side menu. Add the steps of the purification in the following order: equilibration, sample application, column wash, and elution.
	<ol>
		<li>Under equilibration, set the equilibration volume to 10 CV. Ensure that the buffer setting is kept at 0% B.</li>
		<li>Under the sample application tab, set the sample volume by observing the sample loop. The position of the sliding seal indicates the sample volume.</li>
		<li>Under column wash, set the wash volume to 5 CV. Disable fraction collection&#160;for this step; collect the wash as a whole in a beaker during the run, rather than collecting as fractions.</li>
		<li>For elution, set the gradient volume to 14 CV starting from 0% Buffer B to 100% Buffer B.</li>
	</ol>
	</li>
	<li>Save the method and start the run.</li>
	<li>Ensure that the fraction collector starts at the position of the first fraction.</li>
	<li>Enter the run name and click Start.</li>
	<li>During the equilibration phase, monitor the real-time UV curve. Ensure that all connections are secure with no leakage; confirm a complete circuit by looking for the buffer flowing into the waste container. If the UV curve has not stabilized during the last minutes of equilibration, extend the equilibration step until stabilization is achieved (Figure 6) .</li>
	<li>At the time of sample application, switch the tubes going into the waste container to a clean beaker. During this step, collect the unbound proteins flowing out of the waste tubes as the flowthrough (label as FT).</li>
	<li>Look for a sharp increase in the UV curve that likely reaches the maximum possible reading of 3,000 maU (Figure 6). 
	NOTE: A typical curve plateaus at 3,000 maU through most of the sample application step.</li>
	<li>During column wash, as residual unbound proteins are being washed off with only bound proteins remaining on the resin, look for a sharp drop in the UV curve (Figure 6). If the UV curve has not dropped by the end of the wash step, extend the step by clicking hold step until the UV has dropped sufficiently. At the beginning of this step, transfer the waste tubes from the FT beaker to another clean beaker labeled Wash.</li>
	<li>When the elution step starts, ensure that the fraction collector moves into position and begin collecting fractions. Visualize the elution of protein by looking for peaks in the UV curve (Figure 6).</li>
	<li>After run completion, navigate to the Home tab and select Open Run. Open the run to view the chromatogram and select peak integration on the top menu to mark the peaks obtained during the run.</li>
	<li>Select the Fractions tab to visualize the fractions containing eluted protein.</li>
</ol>
7. SDS PAGE analysis
<ol>
	<li>Mix 15 &#181;L of each fraction containing protein with 5 &#181;L of SDS sample loading buffer (Table 1). Heat the samples at 95 &#176;C for 5 min.</li>
	<li>Load samples into precast gels and run at 160 V for 45 min along with prestained protein marker.</li>
	<li>Stain the gels in Coomassie brilliant blue staining solution for 30 min (Table 1).</li>
	<li>Wash the gel in ddH2O (repeat 3x).</li>
	<li>Destain the gel in destaining solution for 1 h (Table 1).</li>
	<li>Observe protein bands; detect FEN1 at the 42 kDa mark.</li>
	<li>Pool fractions containing FEN1 and concentrate the pool using a 10 kDa centrifugal filter. Aliquot and store in the -80&#730; &#176;C freezer.</li>
</ol>

Optimizing FPLC for Efficient Purification of His-Tagged FEN1 Protein

<ol><li>Balakrishnan, L., Bambara, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flap+endonuclease+1%5BTitle%5D)+AND+%22Annu+Rev+Biochem%22%5BJournal%5D)">Flap endonuclease 1.</a> Annu Rev Biochem. 82, 119-138 (2013).</li>
<li>Asagoshi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FEN1+functions+in+long+patch+base+excision+repair+under+conditions+of+oxidative+stress+in+vertebrate+cells%5BTitle%5D)+AND+%22Mol+Cancer+Res%22%5BJournal%5D)">FEN1 functions in long patch base excision repair under conditions of oxidative stress in vertebrate cells.</a> Mol Cancer Res. 8 (2), 204-215 (2010).</li>
<li>Lyamichev, V., Brow, M. A., Dahlberg, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure-specific+endonucleolytic+cleavage+of+nucleic+acids+by+eubacterial+DNA+polymerases%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases.</a> Science. 260 (5109), 778-783 (1993).</li>
<li>Bornarth, C. J., Ranalli, T. A., Henricksen, L. A., Wahl, A. F., Bambara, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+flap+modifications+on+human+FEN1+cleavage%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Effect of flap modifications on human FEN1 cleavage.</a> Biochemistry. 38 (40), 13347-13354 (1999).</li>
<li>Xu, Y., Potapova, O., Leschziner, A. E., Grindley, N. D., Joyce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contacts+between+the+5%27+nuclease+of+DNA+polymerase+I+and+its+DNA+substrate%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Contacts between the 5' nuclease of DNA polymerase I and its DNA substrate.</a> J Biol Chem. 276 (32), 30167-30177 (2001).</li>
<li>Wen-Hui, K., Kuo, K., Chase, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploiting+the+interactions+between+poly-histidine+fusion+tagsand+immobilized+metal+ions%5BTitle%5D)+AND+%22Biotechnol+Lett%22%5BJournal%5D)">Exploiting the interactions between poly-histidine fusion tagsand immobilized metal ions.</a> Biotechnol Lett. 33 (6), 1075-1084 (2011).</li>
<li>Charlton, A., Zachariou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immobilized+metal+ion+affinity+chromatography+of+native+proteins%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Immobilized metal ion affinity chromatography of native proteins.</a> Methods Mol Biol. 421, 25-35 (2008).</li>
<li>Ononye, O. E., Njeri, C. W., Balakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+DNA+processing+enzyme+FEN1+and+its+regulation+by+protein+lysine+acetylation%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Analysis of DNA processing enzyme FEN1 and its regulation by protein lysine acetylation.</a> Methods Mol Biol. 1983, 207-224 (2019).</li>
<li>Econo-column flow adaptor instruction manual. Bio-Rad. , Available from: 
 <a target="_blank" href="https://www.bio-rad.com/webroot/web/pdf/lsr/literature/M7380014.pdf">https://www.bio-rad.com/webroot/web/pdf/lsr/literature/M7380014.pdf</a> (2015).</li>
<li>NGC Chromatography systems and ChromLab software instrument guide version 3.3. Bio-Rad. , Available from: 
 <a target="_blank" href="https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10026252.pdf">https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10026252.pdf</a> (2015).</li>
</ol>

The authors have no competing financial interests or other conflicts of interest.

Affinity chromatography is a widely used technique to purify DNA-binding proteins. Immobilized metal affinity chromatography (IMAC) is a specific type of affinity chromatography that uses metal ions to capture the histidine residues of a peptide sequence. This is why the &#34;6X-His tag&#34; or &#34;poly-His tag&#34; is attached to the N-terminus or the C-terminus of proteins to be purified. Nickel and cobalt are the most commonly used metal ions and vary in their compatibility with reagents such as BME and DTT normally ...

Numerous strategies are available for comprehending cellular biology. One approach involves a top-down strategy, wherein genetic mutations are introduced into a gene, followed by the evaluation of resulting phenotypic changes in a model organism. Conversely, a reductionist approach entails the initial elucidation of molecular mechanisms and enzymatic functions of a particular protein, accompanied by the characterization of its interactions with other cellular components. Subsequently, the impact of this protein on a biological pathway is assessed. Although each research approach possesses its inherent advantages and limitations, achieving a comprehensive understanding of a biological pathway necessitates interdisciplinary investigations.
With DNA being the genetic blueprint of life, understanding the mechanisms of DNA duplication and genome maintenance has been an area of active interest for over seven decades. Studies in the field of DNA replication have yielded copious data concerning the individual structures and functions of numerous replication proteins. These inquiries, which encompass mechanistic aspects and biochemical activity assays, have been made feasible through the purification of these proteins, enabling their meticulous examination in an in vitro milieu. Consequently, protein purification emerges as an indispensable and ubiquitous technique in the majority of research endeavors geared toward unraveling mechanistic insights into DNA replication.
This article presents a methodology for isolating a DNA replication protein tagged with 6X-histidine, which has been overexpressed in bacterial cells. The protein of interest is human flap endonuclease 1 (FEN1), a structure-specific nuclease that plays a pivotal role in lagging strand replication and is also a critical participant in DNA repair pathways like base excision repair (BER)1,2,3. FEN1&#39;s primary function is to cleave at the base of a 5&#39;-displaced flap structure, an intermediate that arises during DNA replication or BER. Initially, biochemical investigations assessing the enzymatic activity of FEN1 suggested a &#34;tracking&#34; mechanism, wherein the nuclease would recognize the free 5&#39;-phosphate end of a flap structure and then follow along the flap to its base before cleaving it4. Subsequent research revealed that FEN1 operates via a &#34;threading&#34; mechanism, wherein it first binds to the base of the flap and then threads the free 5&#39; end through its active site prior to cleavage (Figure 1)5. The ability to overexpress and isolate recombinant FEN1 has facilitated these breakthroughs, enabling researchers to employ it in biochemical and structural investigations.
Affinity chromatography is a commonly used separation method to purify DNA. This technique uses the reversible binding affinity of target proteins towards ligands immobilized on a resin to specifically trap the protein of interest. One of the most widely used bio-affinities is the robust interaction between the amino acid, histidine, and metal ions such as nickel and cobalt and hence, can be captured onto a resin charged with Ni2+ or Co2+.
The DNA sequence encoding a string of 6-9 histidine residues (His) is frequently incorporated into the plasmid construct which encodes the protein of interest (at either the N-terminus or C-terminus), tagging the protein with a 6X-His-tag or a poly-His tag. The His-tagged protein can then be easily purified by immobilized metal affinity chromatography (IMAC), a subtype of affinity chromatography whereby metal ions on the resin capture proteins with an affinity tag, which can later be eluted using appropriate elution agents. Transition metal ions such as Ni2+ and Co2+ can be immobilized onto agarose or silica gel matrices derived from N,N,N&#39;-tris-(carboxymethyl)-ethylenediamine or nitrilotriacetic acid (NTA) groups6.
Metal ligands are known to be robust against degradation by physical, chemical, and biological factors and hold this advantage over other types of ligands6,7. Additionally, the His-tag is a relatively small tag and does not significantly impact protein structure or function7. However, in a bacterial expression system, many chromosomally expressed proteins have an affinity towards metal ions and may co-purify with the target protein. Nickel and cobalt are the typical metal ions used in IMAC matrices. The Ni-NTA resin and the TALON cobalt-based resin are commonly used for the purification of His-tagged proteins.
Ni-NTA versus TALON 
The respective metal ions of both Ni-NTA and TALON are immobilized on the resin through NTA ligands. Ni-NTA is thought to have a higher binding capacity, binding up to 100 mg/mL of protein. This can result in a higher protein yield, with the caveat that contaminant proteins may be co-purified. In contrast, the resin has a higher binding specificity towards His-tagged proteins and may be able to produce fractions of higher purity. In this study, we aim to compare the purification efficiency of both resins using the referenced automated fast protein liquid chromatography system, the NGC system (see the Table of Materials).
Buffers and compatibility 
Buffers are required during protein purification for cell lysis, sample preparation, resin equilibration, and elution of the captured protein from the resin. Tris, MOPS, and HEPES buffers up to a concentration of 100 mM are the known compatible buffers for the Ni-NTA resin. Buffers often include reducing agents to prevent the oxidation of protein and protein aggregation. However, above a threshold limit, reducing agents could strip the resin of metal ions. The recommended concentration of reducing agents such as beta-mercaptoethanol (BME) or dithiothreitol (DTT) is below 1 mM for the above-mentioned nickel- and cobalt-based resins.
The buffers for the purification of hFEN1 are Tris buffers containing NaCl, BME, phenylmethylsulfonyl fluoride (PMSF), EDTA, and glycerol. NaCl maintains the protein in soluble form and disrupts molecular interactions such as DNA binding. BME reduces oxidized proteins and thereby prevents protein aggregation. PMSF) is a protease inhibitor that prevents protease-mediated degradation of target protein. EDTA eliminates divalent cations from the sample, preventing their access to nucleases and proteases. Glycerol enhances the stability of the protein in aqueous form. Additionally, the lysis buffer contains complete protease inhibitor tablets to ensure maximum protection of target protein from degrading proteases during cell lysis. The equilibration and elution buffers contain imidazole, with the elution buffer containing higher quantities for the imidazole to displace the bound protein from the resin during elution.
Next Generation Chromatography (NGC) system 
This automated, medium-pressure chromatography system designed for fast-flow protein liquid chromatography (FPLC) uses two pumps to simultaneously pump two different buffers and is capable of injecting a wide range of sample volumes from 250 &#181;L to 100 mL. The sample loop (known as the Dynaloop in this system), makes it possible to inject larger sample volumes. The system can be operated using the Chromlab software, which facilitates customized method creation, manipulation of purification runs, and analysis of UV peaks and protein fractions.

<table><tbody><tr><td>4x Laemmli sample buffer</td><td>BioRad</td><td>1610747</td><td></td></tr><tr><td>Acetic acid</td><td>Merck</td><td>UN2789</td><td></td></tr><tr><td>Beta-mercaptoethanol (BME)</td><td>Sigma</td><td>M-6250</td><td></td></tr><tr><td>Chromlab software version 6.1.27.0</td><td>BioRad</td><td></td><td>operates the NGC system</td></tr><tr><td>Complete MINI protease inhibitor tablet</td><td>Roche</td><td>11836153001</td><td></td></tr><tr><td>Coomassie Brilliant Blue R</td><td>Sigma</td><td>B0149</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Dot Scientific</td><td>DSD11000</td><td></td></tr><tr><td>Econo-Column glass</td><td>BioRad</td><td>7371512</td><td></td></tr><tr><td>Ethylene diamine tetraacetic acid (EDTA)</td><td>Dot Scientific</td><td>DSE57020</td><td></td></tr><tr><td>Flow adaptor</td><td>BioRad</td><td>7380014</td><td></td></tr><tr><td>Glycerol</td><td>Dot Scientific</td><td>DSG22020</td><td></td></tr><tr><td>Imidazole</td><td>Dot Scientific</td><td>DSI52000</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412</td><td></td></tr><tr><td>Mini PROTEAN TGX gels</td><td>BioRad</td><td>4561084</td><td></td></tr><tr><td>NGC Chromatography System</td><td>BioRad</td><td></td><td>automated liquid chromatography system&amp;nbsp;</td></tr><tr><td>Ni-NTA Agarose</td><td>Qiagen</td><td>1018244</td><td></td></tr><tr><td>Phenyl-methyl-sulfonyl fluoride</td><td>Dot Scientific</td><td>DSP20270</td><td></td></tr><tr><td>PreScission Plus Protein Dual Color Standards&amp;nbsp;</td><td>BioRad</td><td>1610374</td><td></td></tr><tr><td>Sodium chloride</td><td>Dot Scientific</td><td>DSS23020</td><td></td></tr><tr><td>TALON metal affinity resin</td><td>Takara</td><td>635502</td><td></td></tr><tr><td>Tris Base</td><td></td><td>DST60040</td><td></td></tr></tbody></table>

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.

BL21 (DE3) cell lysates expressing hFEN1 were passed through equilibrated Ni-NTA and TALON resins. The Ni-NTA resin is charged with Ni2+ ions and has a high binding capacity. The results show that the Ni-NTA resin yields a higher quantity of FEN1 compared to the TALON resin (Figure 7). The Ni-NTA resin is also known to non-specifically bind to other chromosomally expressed proteins. Cell lysate passed through the cobalt-based resin, was purified with high purity but a lower yield,...

Read this Scientific Article Protocol about Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein at JoVE.com

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.

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. ...

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Biochemistry

2.3K Views.  Indiana University Indianapolis. 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. 

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

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

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 ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

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 ...

Biology

High-throughput Purification of Affinity-tagged Recombinant Proteins

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

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

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

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

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

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

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

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
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&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
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Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
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To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
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Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

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In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
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Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
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You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Size Exclusion and Affinity Chromatography

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by ...

Education

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Chemistry

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

Source: Karch, C. P. et al., <a href="https://app.jove.com/v/60103">Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation.</a>&#160;J. Vis. Exp. (2019)
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.

1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)

	Mix 95 mL of ...

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Immunology

Immunotherapy

Immunogenics and Vaccine Development

Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Source: Ma, Z., et al. <a href="https://app.jove.com/v/55236">Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.</a> J. Vis. Exp. (2017).
This video demonstrates tandem affinity purification &#8212; a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).
This video demonstrates tandem ...