Name: benchtop immobilized metal affinity chromatography, reconstitution and assay of a polyhistidine tagged metalloenzyme for the undergraduate laboratory
Uploaded: 2018-08-23T13:00:00.000+00:00
Duration: 8 min 2 s
Description: 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.

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.

<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://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

Research

JoVE Journal

Biochemistry

17.2K 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

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

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

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

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 ...

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 ...