This publication is based upon work supported by the National Science Foundation under Grant No.&#160; CHE 1708237.&#160;

1. Preparation for Purification
<ol>
	<li>Preparation of the cell-free crude extract
	<ol>
		<li>Obtain a ~9-10 g cell pellet of E. coli (BL21) that overexpressed the polyhistidine tagged metalloprotein25 in a 50 mL conical tube.</li>
		<li>Add 5 mL per gram of room temp lysis/bind buffer (50 mM Phosphate, 300 mM NaCl, 10 mM imidazole at pH 8) to the ~9-10 g of cell pellet for a total volume ~45-50 mL. Periodically vortex to encourage dissolution. If the pellet was frozen, thaw in a tepid water bath.</li>
		<li>Using ice-water bath, chill the cell suspension to ~3 &#176;C in preparation for cell lysis. 
		Note: At this point, cell lysis by sonication, bead milling or another method is possible. Bead milling is demonstrated here because it is rapid, relatively inexpensive, and does not require ear protection.</li>
		<li>Assemble a 50 mL bead milling chamber according to the manufacturer&#8217;s instructions.</li>
		<li>Fill the bead mill chamber half full of chilled 0.1 mm glass beads that have been stored at -20 &#176;C.</li>
		<li>Fill the rest of the chamber with the chilled cell suspension and use 4 &#176;C lysis/bind&#160;buffer to fill the chamber completely.</li>
		<li>Use a small spatula to rotate the shaft of the bead mill and mix the cell suspension with the beads.</li>
		<li>Remove any large bubbles with a pipet, and then screw the lid on.</li>
		<li>Dry off the chamber from any leakage.</li>
		<li>Add about 1 cup of ice to the clear, plastic jacket, invert the filled 50 mL chamber into the ice filled jacket, and screw closed.</li>
		<li>Secure the ice-jacketed chamber onto the motor.</li>
		<li>Turn the bead mill motor on for 15 seconds at 15,800 rpm, then allow to rest for 45 seconds. Repeat 8 times.</li>
		<li>When the 8 cycles are complete, decant the entire cell lysis suspension into a 50 mL or larger tube rated for high speed centrifugation (25,000 x g or higher). Place a pair of balanced tubes in centrifuge and spin at 25,000 x g for 40-45 minutes at 4&#176;C to pellet cell debris and glass beads. 
		Note: If 50 mL or larger tubes rated for high speed centrifugation are not available, remove the glass beads by centrifuging at 1000 - 2000 x g for 2-3 min in a 50 mL conical tube to yield a smaller volume of cell suspension (~25 mL) that can be decanted into a smaller volume tube rated for high speed centrifugation.</li>
		<li>When centrifugation is complete, decant the clear, yellow, cell-free, crude extracts into a clean 50 mL conical tube. Take care not to transfer any cell debris.</li>
		<li>Collect a small sample of the cell-free crude extracts for later analysis of the purification by SDS-PAGE26.</li>
	</ol>
	</li>
	<li>Preparation of the IMAC column
	<ol>
		<li>Obtain a 1.5 x 20 cm column equipped with a lower bed support to retain resin particles, a Luer lock outlet and an upper cap that also contains a Luer lock fitting. Fit the Luer-lock outlet with a stopcock to control flow.</li>
		<li>Working at the benchtop, securely mount the column on a ring stand.</li>
		<li>Obtain a 50% slurry of nickel-bound nitriloacetic acid (Ni-NTA) resin in 20% ethanol that has been stored at 4 &#176;C.</li>
		<li>Gently swirl the bottle to evenly re-suspend the resin.</li>
		<li>Working at room-temperature and on the benchtop, use a graduated pipet to withdraw 2 mL of the slurry, which will yield 1 mL of settled resin capable of binding 50-60 mg of polyhistidine tagged protein, and transfer the slurry to the column.</li>
		<li>Open the stopcock and allow the excess storage solution to drain by gravity from the resin.</li>
		<li>Close the stopcock securely</li>
		<li>Using a Pasteur pipet, carefully add chilled lysis/bind buffer (50 mM Phosphate, 300 mM NaCl, 10 mM imidazole at pH 8) and to the column of resin &#8211; at least 30 mL. Take care not to disturb the resin surface. Run the buffer down the walls of the column to prevent splashing.</li>
		<li>Equilibrate the resin by allowing the lysis/bind buffer to drain slowly from the column by gravity into a collection beaker.</li>
		<li>When the lysis/bind buffer has mostly drained, close the stopcock to stop the flow of buffer when ~5 mL of lysis buffer remains above the resin and leave the column, mounted upright, until ready to proceed or for up to 1 week.</li>
	</ol>
	</li>
</ol>
2. Purification of Polyhistidine-tagged Target by IMAC
<ol>
	<li>Prepare wash buffer (50 mM Phosphate, 300 mM NaCl, 20 mM imidazole pH 8) and elution buffer (50 mM Phosphate, 300 mM NaCl, 250 mM imidazole, 10% glycerol pH 8). Chill at 4&#176;C.</li>
	<li>Prepare the Ni-NTA column for the addition of the cell-free crude extracts by opening the stopcock and allowing remaining lysis/binding buffer to drain by gravity through the Ni-NTA resin. When all the buffer has drained, and the resin surface is exposed, close the stopcock.</li>
	<li>Using a Pasteur pipet, carefully pipet the cell free crude extracts onto the resin, taking care not to disturb the surface. Run the crude extracts down the walls of the column to prevent splashing. The volume of crude extracts applied is dependent on the level of expression of the polyhistidine-tagged protein; ensure the total volume of crude extracts contains 50-60 mg or less of the target protein per mL of Ni-NTA resin (see step 1.2.4).</li>
	<li>When all of the crude extracts have been transferred to the column, open the stopcock so the column can drain by gravity. This flow through is composed of proteins that did not bind to the resin &#8211; collect 100 &#956;L for later analysis of the purification by SDS-PAGE.26</li>
	<li>The viscosity of the cell-free crude extracts can slow the gravity flow of the column considerably. To increase the rate of flow, apply a simple &#8220;hand-pump&#8221;:
	<ol>
		<li>Take a 10 mL&#160;Luer-lock syringe and connect it to the cap of the column using a short length of plastic tubing and Luer-lock fittings between to the column cap and the syringe. Draw out the syringe plunger, and then tightly fit the column cap on the top of the column.</li>
		<li>Gently compress the plunger of the syringe while manually assessing the flow rate by collecting the eluent into a graduated cylinder; rates of 1-2 mL per minute are compatible with the resin and this setup. Gently increase compression of the syringe plunger as the flow rate slows.</li>
		<li>To prevent the introduction of air into the resin, remove the column cap and attached hand-pump when the meniscus of the applied liquid reaches 5-10 mm above the resin bed, and allow the remaining volume to drain by gravity.</li>
	</ol>
	</li>
	<li>When the cell free crude extracts have finished draining and the resin surface is exposed again, use a Pasteur pipet to carefully add ~30 mL of chilled Wash buffer to the column &#8211; taking care not to disturb the surface of the resin. Run the buffer down the walls of the column to prevent splashing.</li>
	<li>Open the stopcock and allow the wash buffer to drain through the column. This process is removing weakly bound proteins from the resin. Discard the eluent.</li>
	<li>While the wash buffer is draining, prepare sixteen, labeled, microcentrifuge tubes for collecting 1 mL fractions.</li>
	<li>When the wash buffer has drained, and the resin surface is exposed again, close the stopcock. Use a Pasteur pipet to carefully add ~30 mL of chilled elution buffer to the column &#8211; taking care not to disturb the surface of the resin. Run the buffer down the walls of the column to prevent splashing.</li>
	<li>Open the stopcock slowly and allow the elution buffer to elute the polyhistidine tagged protein from the column. Collect 1 mL of eluent in each of the marked microcentrifuge tubes, 1 through 16.</li>
	<li>Test the fractions for protein using a colorimetric protein assay such as the Bradford assay or UV-Visible spectroscopy according to the methods specific by the reagent and/or instrument manufacturers27,28. As shown here, the colorimetric assay using Coomassie blue is scaled down to a 100 &#956;L volume in order to sacrifice only a small volume from each fraction.</li>
	<li>Mix 3 &#956;L of each fraction with 100 &#181;L of a colorimetric protein assay reagent and manually observe the formation of any color to indicate the presence of protein in the fraction; detection with a spectrometer is not necessary.</li>
	<li>If protein is found in fraction 16, continue eluting 1 mL fractions and assay for protein periodically, until the eluted fractions no longer contain a significant amount of protein.</li>
	<li>Combine protein-containing fractions into a clean conical tube, and withdraw a 100 &#956;L sample for later analysis by SDS-PAGE26.</li>
	<li>Proceed with reconstitution of the metal ion cofactor or freeze the protein in 3 mL aliquots at -80&#176;C. Ensure that 10% glycerol is included in the elution buffer is a cryoprotectant to stabilize the pure protein during freezing.</li>
	<li>When elution of the Ni-NTA column is complete, that is, all the protein is off the column and collected in fractions, pass another 25 mL of elution buffer through the column, and store the column at 4 &#176;C in ~5 mL of elution buffer for short-term storage, or lysis/bind buffer with 20% ethanol for longer-term storage.</li>
</ol>
3. Reconstitution of Target Protein with Fe2+
<ol>
	<li>Addition of Fe2+ 
	Note: A non-heme iron (II) binding enzyme, such as the L-DOPA dioxygenase in this example, requires an Fe2+ ion for activity; however, the iron (II) readily oxidizes to iron (III), which is inactive,&#160;and a fraction of the protein purifies without any iron at all.
	<ol>
		<li>To begin the reconstitution of the metal, obtain 3 mL of the purified enzyme suspended in elution buffer. If frozen, thaw rapidly using a tepid water bath, and once thawed, put the protein on ice.</li>
		<li>Using the volume of protein in the tube, calculate the amount of sodium ascorbate (198.1 g/mol) necessary to make the final concentration 12.5 mM in the sample. Also, calculate the amount of dithiothreitol (DTT, 154.25 g/mol) necessary to make the final concentration 12.5 mM in the sample.</li>
		<li>Add the solid sodium ascorbate and DTT and gently, but thoroughly, mix to dissolve completely. Take care not to cause protein precipitation with overly aggressive mixing. Add a small amount of iron (II) sulfate heptahydrate (MW 278.01 g/mol) to the protein sample.</li>
		<li>In order to obtain a small enough quantity of the iron salt, tap a few 1 mm granules of FeSO4&#8226;7H2O solid out on a piece of weigh paper, fold the paper over the granule, and crush it with the flat side of a metal spatula.</li>
		<li>Add a grain of the resulting powder to the tube of protein.</li>
		<li>Vortex to mix and a rosy pink color will appear in the tube.</li>
		<li>Incubate the pink solution of protein, capped, for 10-30 minutes on ice. The pink color will slowly fade over time.</li>
	</ol>
	</li>
	<li>Gel filtration
	<ol>
		<li>Use a gel filtration column packed with 10 mL of spherical polyacrylamide gel with a molecular weight exclusion limit of approximately 6,000 and a hydrated particle size range of 90&#8211;180 &#181;m in a 1.5 x 12 cm column, which is also equipped with a 10 mL reservoir. Ensure the column is equipped with lower and upper bed supports in the form of 30 um porous polyethylene disks to retain the resin in the column and prevent the resin bed from running dry. Mount the gel filtration column to a ring stand.</li>
		<li>If using a pre-packaged gel filtration column, remove the cap and pour off the excess buffer above the upper bed support.</li>
		<li>To equilibrate the column, begin by filling the reservoir with buffer compatible with subsequent assay and storage, for example, 50 mM NaH2PO4, 200 mM NaCl, 10% glycerol at pH 8.0.</li>
		<li>If using a pre-packaged gel filtration column, snap off the bottom tip of gel filtration column to start the flow of buffer and expose the Luer slip fitting. Fit a stopcock to the Luer slip end of the column, but leave it open and allow the column to drip by gravity.</li>
		<li>When the buffer has drained from the column and the upper bed support is exposed, close the stopcock, and add the ~3 mL of the Fe (II) reconstituted metalloenzyme to the column by pipetting the solution onto the exposed upper bed support.</li>
		<li>Open the stopcock and allow the entire 3.0 mL sample to enter the column by gravity. Discard these first 3 mL of eluent. Any pink color that forms while handling the solution will be trapped at the top of the column.</li>
		<li>When the column has stopped dripping and the upper bed support is exposed, add 4 mL of the gel-filtration buffer (e.g., 50 mM NaH2PO4, 200 mM NaCl, 10% glycerol at pH 8.0) to the&#160;column. Open the stopcock and collect the eluent into microcentrifuge tubes over eight 0.5 mL fractions.</li>
		<li>Test the fractions for protein using a colorimetric protein assay or UV-Vis27,28 as described earlier.</li>
		<li>Combine protein-containing fractions.</li>
		<li>Withdraw a sample for SDS-PAGE analysis26.</li>
		<li>Proceed with assay or storage of the sample.</li>
	</ol>
	</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Hydroxyphenyl)pyruvate+Dioxygenase+from+Streptomyces+avermitilis:+The+Basis+for+Ordered+Substrate+Addition.">4-Hydroxyphenyl)pyruvate Dioxygenase from Streptomyces avermitilis: The Basis for Ordered Substrate Addition.</a> Biochemistry. 42 (7), 2072-2080 (2003).</li><li>Bugg, T. D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overproduction,+purification+and+properties+of+2,3-dihydroxyphenylpropionate+1,2-dioxygenase+from+Escherichiacoli.+Biochimica+et+Biophysica+Acta+(BBA).">Overproduction, purification and properties of 2,3-dihydroxyphenylpropionate 1,2-dioxygenase from Escherichiacoli. Biochimica et Biophysica Acta (BBA).</a> Protein Structure and Molecular Enzymology. 1202 (2), 258-264 (1993).</li><li>Mendel, S., Arndt, A., Bugg, T. D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-Base+Catalysis+in+the+Extradiol+Catechol+Dioxygenase+Reaction+Mechanism+Site-Directed+Mutagenesis+of+His-115+and+His-179+in+Escherichia+coli+2,3-Dihydroxyphenylpropionate+1,2-Dioxygenase+(MhpB).">Acid-Base Catalysis in the Extradiol Catechol Dioxygenase Reaction Mechanism Site-Directed Mutagenesis of His-115 and His-179 in Escherichia coli 2,3-Dihydroxyphenylpropionate 1,2-Dioxygenase (MhpB).</a> Biochemistry. 43 (42), 13390-13396 (2004).</li><li>Viggiani, A., Siani, L., Notomista, E., Birolo, L., Pucci, P., Di Donato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Conserved+Residues+His-246,+His-199,+and+Tyr-255+in+the+Catalysis+of+Catechol+2,3-Dioxygenase+from+Pseudomonas+stutzeri+OX1.">The Role of the Conserved Residues His-246, His-199, and Tyr-255 in the Catalysis of Catechol 2,3-Dioxygenase from Pseudomonas stutzeri OX1.</a> Journal of Biological Chemistry. 279 (47), 48630-48639 (2004).</li><li>Uragami, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structures+of+substrate+free+and+complex+forms+of+reactivated+BphC,+an+extradiol+type+ring-cleavage+dioxygenase.">Crystal structures of substrate free and complex forms of reactivated BphC, an extradiol type ring-cleavage dioxygenase.</a> Journal of Inorganic Biochemistry. 83 (4), 269-279 (2001).</li><li>Veldhuizen, E. J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steady-state+kinetics+and+inhibition+of+anaerobically+purified+human+homogentisate+1,2-dioxygenase.">Steady-state kinetics and inhibition of anaerobically purified human homogentisate 1,2-dioxygenase.</a> Biochemical Journal. 386 (Pt 2), 305-314 (2005).</li></ol>

The authors have no disclosures.

While the addition of polyhistidine tags to recombinant proteins and their purification by IMAC has become virtually ubiquitous in the biochemical literature3,4,5, applications of IMAC to enzyme purification in the biochemistry teaching laboratory remain sparse, and published methods do not always consider the resource limitations of the teaching laboratory20. Furthermore, the use of IMAC in the teaching laboratory is most effective when coupled to experiments that assess activity and purity, making IMAC purification of an enzyme an ideal instructional activity. In order to extend the application of IMAC to the purification of enzymes, including metalloenzymes, in the teaching laboratory, reliable and inexpensive methods are needed. In this protocol, we demonstrate benchtop IMAC using readily available and inexpensive laboratory supplies, while also addressing the limitations in the application of IMAC to metalloproteins22, by reconstituting the iron(II) dependent metalloenzyme, L-DOPA dioxygenase, post purification. Using the reagents and materials described , we estimate the cost of consumables for eight student groups is between $500-600 per semester to run this protocol, including the analysis steps outlined in Figure 1 and Figure 2.
Due to the ease with which Fe2+ can dissociate from amino acid ligands24 and the facile oxidation of Fe2+ by O2 to Fe3+, reconstitution of non-heme, amino acid-chelated iron(II) into a recombinant metalloenzyme is a typical component of the enzyme purification. When classical chromatography is used, it is possible to avoid total loss of iron in some cases32, but more often, the iron (II) is added back in the presence of reducing agents33,34,35,36 often under an anaerobic atomosphere37,38,39, and in some cases the excess iron is not removed33,34,36, complicating any subsequent assay. Consecutive steps of classical chromatography and an anaerobic atmosphere are not realistic for the undergraduate laboratory, prompting the development of this protocol.
While the manual preparation of the Ni-NTA column and the processing of samples largely by gravity does take additional time and effort when compared to pre-packaged columns and automated chromatography instrumentation, the manual steps allow for hands-on learning by the student that result in increased understanding of the science behind the process. The addition of an iron (II) salt under the conditions outlined here is particularly sensitive to excess dithiothreitol. If a student mistakenly adds an excess of dithiothreitol, a precipitation event is likely. We have found it helpful to require students to perform calculations of reagent quantities before arriving in lab, so laboratory time can be used most effectively at the bench. The entire benchtop IMAC purification - from cell-lysis to protein elution - can be accomplished in one 4-hour laboratory period, followed by reconstitution and assay in a subsequent lab period.

The first reports of the purification of a polyhistidine tagged protein from extracts of a host organism using chelation of the histidine tag by an immobilized metal entered the literature in 19881,2. Since that time, the addition of polyhistidine tags to recombinant proteins and their purification by immobilized metal affinity chromatography (IMAC) have become virtually ubiquitous in the biochemical literature3,4,5. IMAC purification methods can be implemented on the benchtop, using automated chromatography and in spin-column formats. While affinity purification methods, especially IMAC, are widely used in the research laboratory, they are less common in the undergraduate teaching laboratory. The most widely used laboratory textbooks for the biochemistry laboratory do not routinely teach these methods, instead opting for more traditional ion-exchange or dye-binding chromatography6,7,8,9. For example, the purification of lactate dehydrogenase by Anderson10 uses affinity by dye-binding, and the purification of Bovine milk &#945;-lactalbumin7,11 by Boyer uses a nickel-nitriloacetic acid matrix, but no recombinant poly-histidine tag, relying instead on intrinsic affinity of the protein for the resin. Some modern undergraduate laboratory textbooks and publications do implement immobilized metal affinity chromatography on poly-histidine tagged protein targets such as green or red-fluorescent proteins12,13,14,15, antibodies16, and selected enzymes17,18,19,20, even some of unknown function21. Arguably, the purification of an enzyme is preferable in the teaching laboratory, because the target can be assayed for activity in subsequent sessions, enriching the experience of &#34;real science&#34; on the part of the student; indeed, these types of laboratory experiences have been published and beneficial outcomes on student learning reported17,18,20,21. And yet, applications of IMAC to enzyme purification in the biochemistry teaching laboratory remain sparse, and the published methods may even presume access to chromatography instrumentation that is typically unavailable for use in the classroom laboratory20. There are also limitations in the application of IMAC to metalloproteins, especially those which bind redox-sensitive divalent metals that are essential to activity22. Frequently, the metal ion is lost or oxidized during purification yielding an inactive enzyme ill-suited to the undergraduate laboratory.
A full one-third of enzymes bind a metal ion23, and despite a nearly universal requirement for iron in all forms of life23, iron is arguably among the most problematic metal ions in enzymology. Non-heme Fe2+ binding enzymes are particularly prone to loss and/or oxidation of the metal during IMAC; presumably due to the lack of a dedicated organic ligand like heme and the ease with which Fe2+ can dissociate from amino acid ligands24. Furthermore, the oxygen dependent oxidation of Fe2+ to Fe3+ is spontaneous in aqueous solution, due to the negative free energy change and the relative stability of Fe3+. Often, these challenges are overcome by use of anaerobic atmosphere and/or non-IMAC chromatographic methods22. In this article, we will demonstrate the use of benchtop IMAC to purify the Fe2+ dependent metalloenzyme L-DOPA dioxygenase using simple, inexpensive chromatography supplies, followed by the reconstitution of the active site Fe2+, and enzymatic assay. These methods are standard in our own undergraduate biochemistry laboratory of 6-12 student groups and can be used to expand the repertoire of enzyme investigations at the undergraduate level.

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			<td>PI88221</td>
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			<td>737-1522&#160;</td>
			<td>1.5 x 20 cm, 35 ml, 2ea,&#160;</td>
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			<td>7318211</td>
			<td>Pkg of 1, 1.6 mm ID/0.8 mm wall, 10 m, low-pressure tubing for liquid handling</td>
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			<td>23236</td>
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			<td>VWR</td>
			<td>89000-028</td>
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			<td>13-676-10F</td>
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			<td>Bio-Rad</td>
			<td>732-2010</td>
			<td>box of 30</td>
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			<td>Genscript</td>
			<td>MB01015</td>
			<td>5mL (Dilute 1:5 with sample)</td>
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			<td height="21" style="height:21px;">ExpressPlus PAGE Gel, 4-20%, 12 wells</td>
			<td>Genscript</td>
			<td>M42012</td>
			<td>20 gels</td>
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			<td>Fisher</td>
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			<td>case of 500</td>
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			<td>110803-50SS</td>
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			<td>1107900-101</td>
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			<td>Fisher</td>
			<td>3119-0050PK</td>
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			<td>1658004</td>
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			<td style="width:145px;">Bio-Rad</td>
			<td>1645052</td>
			<td style="width:1113px;">100&#8211;120/220&#8211;240 V, power supply for high-current applications, includes power cord</td>
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			<td height="21" style="height:21px;width:596px;">UV-1800 with UV-Probe Software</td>
			<td style="width:145px;">Shimadzu</td>
			<td>UV-1800</td>
			<td style="width:1113px;"></td>
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			<td height="21" style="height:21px;width:596px;">Kintek Global Kinetic Explorer</td>
			<td style="width:145px;">Kintek Corp</td>
			<td>version 6</td>
			<td style="width:1113px;">https://www.kintekexplorer.com/downloads/</td>
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</table>

benchtop immobilized metal affinity chromatography, reconstitution and assay of a polyhistidine tagged metalloenzyme for the undergraduate laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box - equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.

These representative results were collected by undergraduate students as they executed this protocol during two course laboratory periods of BCM 341: Experimental Biochemistry at Muhlenberg College. Figure 1 demonstrates the results of the purification of a 20 kDa poly-histidine tagged metalloenzyme, L-DOPA dioxygenase, as performed by two undergraduate students during a 4-hour classroom laboratory period and analyzed by SDS-PAGE by the same students during a subsequent laboratory period. The poly-histidine tagged protein is effectively purified (Figure 1, lane 2). An immunoblot of the gel using antibody against the poly-histidine tag indicates that a small amount of the poly-histidine tagged target protein is lost in the flow-through (data not shown), likely because the greater than 100 mg quantity of target protein in the lysate exceeded the binding capacity of the resin.
Figure 2 demonstrates student-collected activity data on the enzymatic reaction of the poly-histidine tagged metalloenzyme target, L-DOPA dioxygenase, after reconstitution with iron (II) and subsequent gel-filtration as described by the protocol herein. The five second dead-time before data collected begins is typical of students executing this technique for the first time. The robust activity of the metalloenzyme was detected using a published assay, and yielded steady-state kinetic parameters consistent with published results29. Steady-state kinetic parameters were determined by fitting the progress curves30 shown in Figure 2; however, non-linear least squares fitting of initial rates collected over a series of substrate concentrations is equally possible31.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58012/58012fig1.jpg" /> 
Figure 1. SDS-PAGE26 analysis of the poly-histidine tagged protein before, during and after purification. Lanes 1 - Molecular weight markers, 2-purified protein (20 kDa) post Ni-NTA, 3- Ni-NTA flow through, 4 - cell free crude extracts prior to purification, 5 - cell-debris pellet post lysis. Samples were prepared using 5x sample/Loading buffer and separated on pre-cast 4-20% polyacrylamide gels using a gel electrophoresis system. <a href="https://www.jove.com/files/ftp_upload/58012/58012fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58012/58012fig2.jpg" /> 
Figure 2. Steady state assay of the Iron (II) reconstituted metalloenzyme (i.e., L-DOPA dioxygenase, 10&#181;M) with substrate (L-DOPA - 5 &#181;M (red), 25 &#181;M (green), 50 &#181;M (blue)) in buffer (50 mM Phosphate, 200 mM NaCl, pH 8). Traces depict product formation at 414nm. Absorbance data were continuously acquired using 1 mL methacrylate cuvettes in a split-beam scanning UV-Visible spectrometer29. Raw data are fit to a model of the Michaelis-Menten steady-state approximation (KM 30.8 &#181;M &#177; 14.4, kcatapparent 2.3 s-1 &#177; 0.05)30. <a href="https://www.jove.com/files/ftp_upload/58012/58012fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory at JoVE.

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

benchtop-immobilized-metal-affinity-chromatography-reconstitution

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

17.1K Views.  Muhlenberg College. Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Video: Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

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 ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known small molecule-protein interactions and to find potential protein targets for natural products. Compared with other methods, DARTS uses native, unmodified, small molecules and is simple and easy to operate. In this study, we further enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The protein-ligand interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used the mTOR-rapamycin interaction as an exemplary case for establishment of our protocol. From the proteolytic curve we saw that the proteolysis of mTOR by pronase was inhibited by the presence of rapamycin. The dose-dependency curve allowed us to estimate the binding affinity of rapamycin and mTOR. This method is likely to be a powerful and simple method for accurately identifying novel target proteins and for the optimization of drug target engagement.

A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...