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>

<ol>
	<li>Hochuli, E., Bannwarth, W., D&#246;beli, H., Gentz, R., St&#252;ber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Approach+to+Facilitate+Purification+of+Recombinant+Proteins+with+a+Novel+Metal+Chelate+Adsorbent.">Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent.</a> Nature Biotechnology. 6 (11), 1321 (1988).</li><li>Smith, M. C., Furman, T. C., Ingolia, T. D., Pidgeon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chelating+peptide-immobilized+metal+ion+affinity+chromatography.+A+new+concept+in+affinity+chromatography+for+recombinant+proteins.">Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins.</a> Journal of Biological Chemistry. 263 (15), 7211-7215 (1988).</li><li>Block, H., 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=Chapter+27+Immobilized-Metal+Affinity+Chromatography+(IMAC):+A+Review.">Chapter 27 Immobilized-Metal Affinity Chromatography (IMAC): A Review.</a> Methods in Enzymology. , 439-473 (2009).</li><li>Derewenda, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+recombinant+methods+and+molecular+engineering+in+protein+crystallization.">The use of recombinant methods and molecular engineering in protein crystallization.</a> Methods. 34 (3), 354-363 (2004).</li><li>Gr&#228;slund, S., 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=Protein+production+and+purification.">Protein production and purification.</a> Nature Methods. 5 (2), 135-146 (2008).</li><li>Switzer, R. L., Garrity, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental Biochemistry. , (1999).</li><li>Boyer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern Experimental Biochemistry, 3rd Edition. , (2001).</li><li>le Maire, M., Chabaud, R., Herv&#233;, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Guide to Biochemistry, Enzymology, and Protein Physical Chemistry: A Study of Aspartate Transcarbamylase. , (2012).</li><li>Bettelheim, F. A., Landesburg, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Experiments for General, Organic, and Biochemistry. , (2013).</li><li>Anderson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+chromatography+of+lactate+dehydrogenase:+An+experiment+for+the+undergraduate+biochemistry+laboratory.">Affinity chromatography of lactate dehydrogenase: An experiment for the undergraduate biochemistry laboratory.</a> Journal of Chemical Education. 65 (10), 901 (1988).</li><li>Boyer, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+milk+whey+&#945;-lactalbumin+by+immobilized+metal-ion+affinity+chromatography.">Purification of milk whey &#945;-lactalbumin by immobilized metal-ion affinity chromatography.</a> Journal of Chemical Education. 68 (5), 430 (1991).</li><li>Moffet, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+gene+mutation+to+protein+characterization.">From gene mutation to protein characterization.</a> Biochemistry and Molecular Biology Education. 37 (2), 110-115 (2009).</li><li>Sommer, C. A., Silva, F. H., Novo, M. T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teaching+molecular+biology+to+undergraduate+biology+students:+An+illustration+of+protein+expression+and+purification*.">Teaching molecular biology to undergraduate biology students: An illustration of protein expression and purification*.</a> Biochemistry and Molecular Biology Education. 32 (1), 7-10 (2004).</li><li>Wu, Y., Zhou, Y., Song, J., Hu, X., Ding, Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+green+and+red+fluorescent+proteins+to+teach+protein+expression,+purification,+and+crystallization.">Using green and red fluorescent proteins to teach protein expression, purification, and crystallization.</a> Biochemistry and Molecular Biology Education. 36 (1), 43-54 (2008).</li><li>Ward, W. W., Swiatek, G. C., Gonzalez, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+in+biotechnology+education.">Green fluorescent protein in biotechnology education.</a> Methods in enzymology. 305, 672-680 (2000).</li><li>Kay, B. K., Winter, J., McCafferty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Phage Display of Peptides and Proteins: A Laboratory Manual. , (1996).</li><li>Arkus, K. A. J., Jez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+protein+chemistry+laboratory.">An integrated protein chemistry laboratory.</a> Biochemistry and Molecular Biology Education. 36 (2), 125-128 (2008).</li><li>Crowley, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+purification,+and+characterization+of+a+recombinant+flavin+reductase+from+the+luminescent+marine+bacterium+Photobacterium+leiognathi.">Expression, purification, and characterization of a recombinant flavin reductase from the luminescent marine bacterium Photobacterium leiognathi.</a> Biochemistry and Molecular Biology Education. 38 (3), 151-160 (2010).</li><li>Colabroy, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+writing-intensive,+methods-based+laboratory+course+for+undergraduates.">A writing-intensive, methods-based laboratory course for undergraduates.</a> Biochemistry and Molecular Biology Education: A Bimonthly Publication of the International Union of Biochemistry and Molecular Biology. 39 (3), 196-203 (2011).</li><li>Kreiling, J. L., Brader, K., Kolar, C., Borgstahl, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+and+hands-on+research+course+in+protein+purification+and+characterization:+Purification+and+crystal+growth+of+human+inosine+triphosphate+pyrophosphatase.">A real-time and hands-on research course in protein purification and characterization: Purification and crystal growth of human inosine triphosphate pyrophosphatase.</a> Biochemistry and Molecular Biology Education. 39 (1), 28-37 (2011).</li><li>Gray, C., 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=Known+structure,+unknown+function:+An+inquiry-based+undergraduate+biochemistry+laboratory+course.">Known structure, unknown function: An inquiry-based undergraduate biochemistry laboratory course.</a> Biochemistry and Molecular Biology Education. 43 (4), 245-262 (2015).</li><li>Kocabas, E., Hernick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloenzymes:+Use+of+Recombinant+Protein+Expression+and+Affinity+Tags+to+Aid+Identification+of+Native+Metal+Ion+Cofactors.">Metalloenzymes: Use of Recombinant Protein Expression and Affinity Tags to Aid Identification of Native Metal Ion Cofactors.</a> Biochemistry &amp; Analytical Biochemistry. 2 (2), 1-3 (2013).</li><li>Ellis, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloenzymes.">Metalloenzymes.</a> Reviews in Cell Biology and Molecular Medicine. , (2006).</li><li>Williams, R. J. P., Begley, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metallo-Enzymes+and+Metallo-Proteins,+Chemistry+of.">Metallo-Enzymes and Metallo-Proteins, Chemistry of.</a> Wiley Encyclopedia of Chemical Biology. , (2007).</li><li>Mierendorf, R. C., Morris, B. B., Hammer, B., Novy, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+Purification+of+Recombinant+Proteins+Using+the+pET+System.">Expression and Purification of Recombinant Proteins Using the pET System.</a> Molecular Diagnosis of Infectious Diseases. , 257-292 (1998).</li><li>He, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laemmli-SDS-PAGE.">Laemmli-SDS-PAGE.</a> BIO-PROTOCOL. 1 (11), (2011).</li><li>He, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bradford+Protein+Assay.">Bradford Protein Assay.</a> BIO-PROTOCOL. 1 (6), (2011).</li><li>Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Quantitation.">Protein Quantitation.</a> Materials and Methods. , (2017).</li><li>Colabroy, K. L., Hackett, W. T., Markham, A. J., Rosenberg, J., Cohen, D. E., Jacobson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+L-DOPA+2,3-dioxygenase,+a+single-domain+type+I+extradiol+dioxygenase+from+lincomycin+biosynthesis.">Biochemical characterization of L-DOPA 2,3-dioxygenase, a single-domain type I extradiol dioxygenase from lincomycin biosynthesis.</a> Arch Biochem Biophys. 479 (2), 131-138 (2008).</li><li>Johnson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitting+enzyme+kinetic+data+with+KinTek+Global+Kinetic+Explorer.">Fitting enzyme kinetic data with KinTek Global Kinetic Explorer.</a> Methods in Enzymology. , 601-626 (2009).</li><li>Kemmer, G., Keller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+least-squares+data+fitting+in+Excel+spreadsheets.">Nonlinear least-squares data fitting in Excel spreadsheets.</a> Nature Protocols. 5 (2), 267-281 (2010).</li><li>Wang, Y. Z., Lipscomb, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning,+overexpression,+and+mutagenesis+of+the+gene+for+homoprotocatechuate+2,3-dioxygenase+from+Brevibacterium+fuscum.">Cloning, overexpression, and mutagenesis of the gene for homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum.</a> Protein Expr Purif. 10 (1), 1-9 (1997).</li><li>Amaya, A. A., Brzezinski, K. T., Farrington, N., Moran, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+human+homogentisate+1,2-dioxygenase.">Kinetic analysis of human homogentisate 1,2-dioxygenase.</a> Archives of Biochemistry and Biophysics. 421 (1), 135-142 (2004).</li><li>Johnson-Winters, K., Purpero, V. M., Kavana, M., Nelson, T., Moran, G. 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.

<table><tbody><tr><td>consumables</td><td></td><td></td><td></td></tr><tr><td>BeadBeater 0.1mm glass beads</td><td>BioSpec Products</td><td>11079101</td><td>1 pound each</td></tr><tr><td>50mL Conical Tubes with Screw Caps, sterile</td><td>VWR</td><td>21008-178</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Bioreagents</td><td>BP358-1</td><td></td></tr><tr><td>Potassium phosphate, monobasic</td><td>Acros (Fisher)</td><td>AC42420-5000</td><td></td></tr><tr><td>Sodium Ascorbate</td><td>Acros (Fisher)</td><td>AC35268-1000&amp;nbsp;</td><td></td></tr><tr><td>DTT (Dithiothreitol)</td><td>Lab Scientific</td><td>D-115</td><td></td></tr><tr><td>Iron(II) sulfate heptahydrate</td><td>Sigmaaldrich</td><td>310077</td><td></td></tr><tr><td>HisPur NiNTA Resin&amp;nbsp;</td><td>Fisher (pierce)</td><td>PI88221</td><td></td></tr><tr><td>Econo-Column Chromatography Columns - 1.5 x 20cm</td><td>Bio-Rad</td><td>737-1522&amp;nbsp;</td><td>1.5 x 20 cm, 35 ml, 2ea,&amp;nbsp;</td></tr><tr><td>Stopcock Valve, one way, female to male luer</td><td>Kimble</td><td>420163-0000</td><td>pack of 50</td></tr><tr><td>BD 10mL luer-loc syringe (non-sterile, without needle)</td><td>VWR</td><td>301029</td><td></td></tr><tr><td>Fitting for tubing: 1.6 mm Barb to Female Luer</td><td>Biorad</td><td>7318222</td><td></td></tr><tr><td>Fitting for tubing: 1.6 mm Barb to Male Luer&amp;nbsp;</td><td>Biorad</td><td>7318225</td><td></td></tr><tr><td>Silicon Tubing (1.6 mm ID/0.8 mm wall, for 0.2-5 ml/min on Peristaltic Pump)</td><td>Bio-Rad</td><td>7318211</td><td>Pkg of 1, 1.6 mm ID/0.8 mm wall, 10 m, low-pressure tubing for liquid handling</td></tr><tr><td>Glycerol</td><td>Fisher (Pierce)</td><td>17904</td><td></td></tr><tr><td>Coomassie Plus (Bradford) Protein Assay</td><td>Thermo Scientitic</td><td>23236</td><td></td></tr><tr><td>Microcentrifuge Tubes, snap cap, 1.5mL</td><td>VWR</td><td>89000-028</td><td></td></tr><tr><td>Fisherbrand polystyrene disposable serological pipets</td><td>Fisher</td><td>13-676-10F</td><td></td></tr><tr><td>Fiserbrand universal pipet pump</td><td>Fisher&amp;nbsp;</td><td>14-955-110</td><td></td></tr><tr><td>Fisherbrand Transfer Pipets</td><td>Fisher&amp;nbsp;</td><td>13-711-9AM</td><td></td></tr><tr><td>Econo-Pac 10DG desalting columns</td><td>Bio-Rad</td><td>732-2010</td><td>box of 30</td></tr><tr><td>ExpressPlus PAGE 5x sample buffer</td><td>Genscript</td><td>MB01015</td><td>5mL (Dilute 1:5 with sample)</td></tr><tr><td>ExpressPlus PAGE Gel, 4-20%, 12 wells</td><td>Genscript</td><td>M42012</td><td>20 gels</td></tr><tr><td>Fisherbrand Disposable Cuvettes, Methyacrylate</td><td>Fisher</td><td>14-955-128&amp;nbsp;</td><td>case of 500</td></tr><tr><td>Cuvette Caps Square Disposable</td><td>Fisher</td><td>14-385-999</td><td></td></tr><tr><td>L-DOPA (3,4-dihydroxyphenylalanine)</td><td>Acros</td><td>D9628-5G</td><td></td></tr><tr><td>Permanent Equipment:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>BeadBeater 50mL chamber&amp;nbsp;</td><td>BioSpec Products</td><td>110803-50SS</td><td>1 chamber</td></tr><tr><td>BeadBeater</td><td>BioSpec Products</td><td>1107900-101</td><td>350 ml polycarbonate chamber, rotor assembly, motor base, ice-water cooling jacket and one pound of glass beads.</td></tr><tr><td>Centrifuge tubes, High-Speed PPCO, 50mL</td><td>Fisher</td><td>3119-0050PK</td><td></td></tr><tr><td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, 4-gel</td><td>Bio-Rad</td><td>1658004</td><td>&amp;nbsp;4-gel vertical electrophoresis system, includes electrode assembly, companion running module, tank, lid with power cables, mini cell buffer dam</td></tr><tr><td>PowerPac HC Power Supply</td><td>Bio-Rad</td><td>1645052</td><td>100&amp;ndash;120/220&amp;ndash;240 V, power supply for high-current applications, includes power cord</td></tr><tr><td>UV-1800 with UV-Probe Software</td><td>Shimadzu</td><td>UV-1800</td><td></td></tr><tr><td>Kintek Global Kinetic Explorer</td><td>Kintek Corp</td><td>version 6</td><td>https://www.kintekexplorer.com/downloads/</td></tr></tbody></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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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 de Cartagena UDEC

Research

JoVE Journal

Biochemistry

17.3K 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

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

Chemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

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

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

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

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as many of the common di-and trivalent cations used in IMAC resins have an inhibitory effect on enzymes. However, the extent of metal leaching and the impact of various eluting and reducing reagents are poorly understood in large part due to the absence of simple and practical transition metal quantification protocols that use equipment typically available in biochemistry labs. To address this problem, we have developed a protocol to quickly quantify the amount of metal contamination in samples prepared using IMAC as a purification step. The method uses hydroxynaphthol blue (HNB) as a colorimetric indicator for metal cation content in a sample solution and UV-Vis spectroscopy as a means to quantify the amount of metal present, into the nanomolar range, based on the change in the HNB spectrum at 647 nm. While metal content in a solution has historically been determined using atomic absorption spectroscopy or inductively coupled plasma techniques, these methods require specialized equipment and training outside the scope of a typical biochemistry laboratory. The method proposed here provides a simple and fast way for biochemists to determine the metal content of samples using existing equipment and knowledge without sacrificing accuracy.

We present an assay for easy quantification of metals introduced to samples prepared using immobilized metal affinity chromatography. The method uses hydroxynaphthol blue as the colorimetric metal indicator and a UV-Vis spectrophotometer as the detector.

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as ...

Characterization of Ternary Complex Formation in PROTACs Using Biophysical Assays

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

Optimizing FPLC for Efficient Purification of His-Tagged FEN1 Protein

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

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

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

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

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

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Immunology and Infection

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Biology

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
<div class="chapter_text" id="script_0">
&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
</div>
<div class="chapter_text" id="script_1">
Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
</div>
<div class="chapter_text" id="script_2">
To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
</div>
<div class="chapter_text" id="script_3">
Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

</div>
<div class="chapter_text" id="script_4">
In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
</div>
<div class="chapter_text" id="script_5">
Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
</div>
<div class="chapter_text" id="script_6">
You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
Thanks for watching!
</div>

Size Exclusion and Affinity Chromatography

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

Education

JoVE Science Education

Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

Source: Pokhrel, A. et al. A <a href="https://jove.unicartagenaproxy.elogim.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018)
In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

Oczyszczanie białek oparte na chromatografii powinowactwa niklu: technika oczyszczania rekombinowanych białek znakowanych polihistydyną z lizatu komórek bakteryjnych

Source: Pokhrel, A. et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018)
In this ...