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 &#...

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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flap+endonuclease+1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FEN1+functions+in+long+patch+base+excision+repair+under+conditions+of+oxidative+stress+in+vertebrate+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-specific+endonucleolytic+cleavage+of+nucleic+acids+by+eubacterial+DNA+polymerases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+flap+modifications+on+human+FEN1+cleavage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contacts+between+the+5'+nuclease+of+DNA+polymerase+I+and+its+DNA+substrate.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+the+interactions+between+poly-histidine+fusion+tagsand+immobilized+metal+ions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilized+metal+ion+affinity+chromatography+of+native+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+DNA+processing+enzyme+FEN1+and+its+regulation+by+protein+lysine+acetylation.">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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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.

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

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

In this study, we aim to purify flap endonuclease 1 or FEN1, a crucial DNA replication protein using affinity chromatography. This will allow us to investigate FEN1's function in DNA replication and repair in a controlled in vitro environment, advancing our understanding of its functions and biological roles. Various types of chromatography are used to purify DNA binding proteins, the most common being affinity chromatography.Within affinity chromatography, immobilized metal affinity chromatography is one of the strongest tools to purify His-tagged proteins. Since the interaction between the His-tagged and the metal ion, usually nickel or cobalt is very strong. Two crucial factors for successful protein purification process are protein yield and protein purity.Depending on the protein's intended use, either yield or purity might be preferred over the other. Our findings show that selecting the appropriate resin for the purification of His-tagged proteins might yield results desirable for the protein's downstream applications.

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

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. ...

affinity-purification-6x-his-tagged-protein-using-fast-protein-liquid

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JoVE Journal

Biochemistry

Affinity Column Preparation and Packing for Fast Protein Liquid Chromatography (FPLC)

To begin, fit the columns to a clamp stand as straight as possible. Plug the bottom of each column with a stopper. Add one to two milliliters of starting buffer to the bottom of each column to prevent air bubbles during column packing.Then open the top of the columns and pour in 10 milliliters of each slurry continuously against a metal spatula or glass rod held at the rim of the column to prevent air bubbles. Remove the stopper from the column and connect the column's bottom to one end of the tubing. Attach the other end of the tubing to the UV port of the FPLC system.Verify that the bed support housing is pulled into the flow adapter body and slide the flow adapter body onto the top of the column. With the cam latch in position one, lower the flow adapter. Then switch the cam latch to position two.Connect the tubing to the system and start the buffer flow for one to two minutes to eliminate air from the tubing and prevent air bubbles during packing. Afterward, switch the latch back to position one, lower the flow adapter smoothly into the buffer, avoiding air bubbles, and switch the latch back to position two. After switching the latch from position two to three, tighten both the cam base and the locking ring.Then, start the buffer flow at 10 milliliters per minute for three minutes or until the resin is well packed.

Priming the Sample Loop of the Fast Protein Liquid Chromatography (FPLC) System for the Purification of His-Tagged FEN1 Protein

After packing the FPLC column, connect the tubing from the top of the sample loop to loop E on the FPLC system. Attach the tubing from the bottom of the sample loop to waste two on the FPLC system. Then insert one end of the tubing into the column port of the FPLC system and place the other end in the waste bottle.Insert the tubing from pump A into filtered double distilled water. Next, open the software. Click on the System Control tab.Double click on the pump settings and select System Pump Inject Loop. Click on the flow rate settings and set the flow rate to 10 milliliters per minute. Enter 0%in the percent B box.Then start the flow to flush the sample loop with water to push the sliding seal downwards. Once the sliding seal reaches the bottom of the sample loop, stop the flow. Then switch pump A from double distilled water to the starting buffer.Connect the tubing from the top of the sample loop to waste two and the tubing from the bottom to loop E.Leave the tubing from the column port in the waste and start the flow again at 10 milliliters per minute.

Purification of His-Tagged FEN1 Protein Using the FPLC System and Its Analysis Using FPLC-Linked Software

To begin, pack the FPLC column and prepare the sample loop for sample loading. Now go to the software. Double-click on the pump settings and select Manual Load Loop.Now connect the tubing from the column port to the tubing on top of the flow adapter. Unscrew the tubing from loop E and place it into the waste container. Then using a 30-milliliter syringe, draw up the sample and attach the syringe to the injection port adapter.Screw the syringe into the injection port and inject the sample, ensuring it is visibly injected into the sample loop. Now plug the injection port with the column cap. Connect the tubing from the bottom of the sample loop to the loop E port.Then place the tubing from pump B into the elution buffer. Navigate to the Home tab and select New Method. After that, select the Method Settings tab on the left column.Go to the Column Type dropdown menu and select Custom. Enter the column volume as five milliliters and the required flow rate for the run as one milliliter per minute. Navigate to unit selection and select CV, column volumes, as the method base unit.Verify that inlet A is set to buffer A1 and inlet B to buffer B1.Then select the Method Outline tab on the left-side menu. Under Equilibration, set the equilibration volume to 10 CV.Under the Sample Application tab, set the sample volume by observing the sample loop. Navigate to Column Wash and set the wash volume to five CV.For Elution, set the gradient volume to 14 CV, starting from 0%buffer B to 100%buffer B.Save the method and start the run.Ensure that the fraction collector starts at the position of the first fraction. Enter the run name and click Start. During sample application, switch the tubes currently going into the waste container to a clean beaker.Collect the unbound proteins flowing out of these tubes as the flow through and label it as FT.After the run is complete, navigate to the Home tab and select Open Run. Open the run to view the chromatogram. Select Peak Integration on the top menu to mark the obtained peaks.Select the Fractions tab to visualize the fractions containing eluted protein.