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/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>

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 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.

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. ...

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

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Biochemistry

1.4K Views.  Indiana University Indianapolis. In deze studie willen we flap endonuclease 1 of FEN1, een cruciaal DNA-replicatie-eiwit, zuiveren met behulp van affiniteitschromatografie. Dit zal ons in staat stellen om de functie van FEN1 bij DNA-replicatie en -herstel in een gecontroleerde in vitro-omgeving te onderzoeken, waardoor ons begrip van de functies en biologische rollen ervan wordt vergroot. Verschillende soorten chromatografie worden gebruikt om DNA-bindende eiwitten te zuiveren, waarvan affin...

Video: Auteur in de schijnwerpers: Optimalisatie van affiniteitschromatografie voor His-tagged FEN1-eiwit

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

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

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