Name: detection and removal of nuclease contamination during purification of recombinant prototype foamy virus integrase
Uploaded: 2017-12-08T14:00:00
Description: Watch this Scientific Journal Video about Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase at JoVE.com

This work was supported by NIH AI099854 and AI126742 to KEY.

1. Induce PFV IN Expression in E. coli
<ol>
	<li>Add 1 &#956;L of PFV IN expression plasmid (10 ng/&#956;L) to 20 &#956;L of E. coli BL21(DE3) pLysS (20 &#956;L aliquot of commercially available cells has &#62;2 x 106 CFU/&#956;g plasmid) in a 1.5 mL tube. Mix gently by finger tapping or flicking the tube. Incubate on ice 5 min.
	<ol>
		<li>Heat shock for exactly 30 s in a 42 &#730;C water bath and then immediately return to ice for 2 min.</li>
		<li>Add 80...

<ol>
	<li>Lopez, M. A., Mackler, R. M., Yoder, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+nuclease+contamination+during+purification+of+recombinant+prototype+foamy+virus+integrase.">Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase.</a> J Virol Methods. 235, 134-138 (2016).</li><li>Senavirathne, G., 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=Widespread+nuclease+contamination+in+commonly+used+oxygen-scavenging+systems.">Widespread nuclease contamination in commonly used oxygen-scavenging systems.</a> Nat Methods. 12 (10), 901-902 (2015).</li><li>Gunn, K. H., Marko, J. F., Mondragon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthogonal+single-molecule+experiment+reveals+multiple-attempt+dynamics+of+type+IA+topoisomerases.">An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases.</a> Nat Struct Mol Biol. 24 (5), 484-490 (2017).</li><li>Valkov, E., 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=Functional+and+structural+characterization+of+the+integrase+from+the+prototype+foamy+virus.">Functional and structural characterization of the integrase from the prototype foamy virus.</a> Nucleic Acids Res. 37 (1), 243-255 (2009).</li><li>Coffin, J. M., Hughes, S. H., Varmus, H. 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> Retroviruses. , (1997).</li><li>Li, M., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+viral+DNA+ends+channels+the+HIV-1+integration+reaction+to+concerted+integration.">Processing of viral DNA ends channels the HIV-1 integration reaction to concerted integration.</a> J Biol Chem. 280 (32), 29334-29339 (2005).</li><li>Li, M., Mizuuchi, M., Burke, T. R., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+DNA+integration:+reaction+pathway+and+critical+intermediates.">Retroviral DNA integration: reaction pathway and critical intermediates.</a> EMBO J. 25 (6), 1295-1304 (2006).</li><li>Sinha, S., Grandgenett, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+immunodeficiency+virus+type+1+integrase+exhibits+a+capacity+for+full-site+integration+in+vitro+that+is+comparable+to+that+of+purified+preintegration+complexes+from+virus-infected+cells.">Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells.</a> J Virol. 79 (13), 8208-8216 (2005).</li><li>Goodarzi, G., Im, G. J., Brackmann, K., Grandgenett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+integration+of+retrovirus-like+DNA+by+human+immunodeficiency+virus+type+1+integrase.">Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.</a> J Virol. 69 (10), 6090-6097 (1995).</li><li>Vora, A. C., Grandgenett, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+catalytic+properties+of+retrovirus+integrase-DNA+complexes+capable+of+efficiently+performing+concerted+integration.">Assembly and catalytic properties of retrovirus integrase-DNA complexes capable of efficiently performing concerted integration.</a> J Virol. 69 (12), 7483-7488 (1995).</li><li>Jones, N. D., 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=Retroviral+intasomes+search+for+a+target+DNA+by+1D+diffusion+which+rarely+results+in+integration.">Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.</a> Nat Commun. 7, 11409 (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), (2012).</li></ol>

Recombinant proteins that interact with DNA, such as DNA repair proteins, oxygen scavengers for single molecule microscopy applications, or retroviral integrases, should be free of contaminating bacterial nucleases2,3. These contaminants may confuse the interpretation of results during bulk biochemical or single molecule assays.
We have found that a bacterial nuclease frequently co-purifies with PFV IN. However, PFV IN displays an affi...

Biochemical and single molecule studies of protein interactions with DNA require exceptionally pure recombinant proteins. Contaminating nucleases from bacteria can obscure the results of these assays. A contaminating nuclease has been found in preparations of recombinant proteins oxygen scavenger protocatechuate-3,4-dioxygenase (PCD) and prototype foamy virus (PFV) integrase (IN) isolated from Escherichia coli (E. coli)1,2,3.
Retroviral integration assays rely on the conversion of supercoiled DNA to nicked or linear products as a measure of IN activity4. During cellular infection IN joins the two ends of a viral cDNA to the host chromatin5. Each end joining reaction is termed strand transfer. Assays of recombinant IN activity may join two DNA oligomers mimicking the viral cDNA ends to a target DNA in a concerted integration reaction4,5,6,7,8. Alternatively recombinant IN may join only one DNA end in a non-physiologically relevant half site integration reaction9,10. When supercoiled plasmid DNA is the target of integration, concerted integration products are linearized DNA and half site integration products are relaxed circles. These reaction products are identified by their relative mobility during agarose gel electrophoresis1. If the recombinant IN has a contaminating nuclease, there will be spurious relaxed circles or a possibly linearized plasmid confusing the experimental results. Viral DNA oligomers may be fluorescently labeled to conclusively identify integration products, as opposed to nuclease products. However, IN greatly favors supercoiled DNA targets; any loss of supercoiled plasmid to relaxed circles or linear DNA by contaminating nuclease could skew results and interpretation of data11. Thus it is imperative to remove bacterial nucleases from retroviral IN preparations.
PFV IN has a different affinity for heparin sepharose compared to the bacterial nuclease1. PFV IN and the nuclease may be separated by a linear gradient elution from heparin sepharose. The nuclease is not readily detected by an ultraviolet (UV) absorbance at 280 nm peak or by analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Instead, the nuclease is detected by a nuclease activity assay employing the conversion of a supercoiled plasmid to relaxed circles or linear products. Each fraction following heparin sepharose chromatography is tested for nuclease activity. PFV IN and the nuclease contaminant have no difference in affinity for Mono-Q anion resin. There is a small difference in affinity for Mono-S cation resin. However, the Mono-S resolution of bacterial nuclease and PFV IN would not allow efficient separation of the proteins. Ultimately, heparin sepharose affinity purification offers the best separation of bacterial nuclease from PFV IN and has the advantage of unlimited load volume.
Testing for contaminating nuclease activity may be adapted to other proteins. The protein of interest will likely have alternative affinity characteristics than PFV IN; the difference in binding characteristics of the protein of interest and the nuclease contaminant must be empirically determined. This methodology for identifying nuclease contamination may be adapted to other resins including Mono-S cation or Mono-Q anion exchange resins. Affinity and ion exchange resins may offer a reliable method to isolate a recombinant protein of interest from contaminating nucleases with no limits on the volume of protein during chromatography.

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			<td height="21" style="height:21px;width:216px;">BL21/DE3 Rosetta E. coli</td>
			<td style="width:180px;">EMD Millipore</td>
			<td style="width:119px;">70956-4</td>
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			<td height="21" style="height:21px;width:216px;">LB broth</td>
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			<td style="width:180px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;">NaCl</td>
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			<td height="21" style="height:21px;">DTT&#160;</td>
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			<td height="21" style="height:21px;">UltraPure EDTA</td>
			<td style="width:180px;">Invitrogen/Gibco</td>
			<td style="width:119px;">15575</td>
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			<td height="25" style="height:25px;">MgSO4</td>
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			<td height="21" style="height:21px;">Ethidium bromide</td>
			<td style="width:180px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">BP1302</td>
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			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:119px;">G37-20</td>
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			<td height="21" style="height:21px;">Ni-NTA Superflow</td>
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			<td style="width:180px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0998-01</td>
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			<td style="width:119px;">71493-3</td>
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detection and removal of nuclease contamination during purification of recombinant prototype foamy virus integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant PFV IN is often contaminated with a bacterial nuclease1. Biochemical integration assays depend on the quantitation of the conversion of supercoiled plasmid DNA to relaxed circles and linear products. The presence of a contaminating nuclease could lead to spurious quantitation of these assays. Expression of PFV IN with a hexahistidine tag is induced in E. coli (Figure 1) and first purified by nickel affinity chromatography (Fig...

Watch this Scientific Journal Video about Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase at JoVE.com

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

The overall goal of this protocol is to identify and remove contaminating nuclease activity during protein purification. This method can help answer key questions in the retrovirology field, such as biochemistry, structure, and dynamics of integration. The main advantage of this technique is that it allows identification and removal of contaminating DNA nuclease activity from a recombinant protein of interest during purification.Begin by thawing one pellet of E.Coli expressing prototype foamy virus integrase on ice. Use a sonicator equipped with a 0.5-inch diameter biohorn with a 0.125-inch diameter tapered microtip to sonicate for 30 seconds at 30%amplitude with the tube on ice. After sonication, transfer the cell sample to a cold ultracentrifuge tube, and spin at 120, 000 times g for 60 minutes at four degrees Celsius.Following centrifugation, there should be an obvious pellet, and the supernatant may have a yellow color. Transfer the supernatant into a cold 50-milliliter conical tube. Next, set up the 110-millimeter long, five-millimeter diameter FPLC column with a 1.5 milliliter nickel-charged resin.First, connect the column to the FPLC instrument, and apply buffer. The instrument should be collecting realtime data of conductivity and UV absorbance at 280 nanometers. The conductivity and UV readings should stabilize.If necessary, continue to flow buffers through the column until readings are stable. Using a superloop, load the protein sample onto the column at a flow rate of 0.15 milliliters per minute with a maximum pressure limit of 0.5 megapascal. Attach the superloop to the AKTA FPLC.After washing the column and eluding the proteins, pick 15-microliter aliquots of the initial flowthrough, wash, and peak 280-nanometer absorbance fractions, and add 15 microliters of 2X SDS-PAGE sample buffer to each sample. Boil the samples for three minutes. Load 10 microliters of each sample onto 10%SDS-PAGE gels, along with four microliters of pre-stained protein standards.Visually inspect the gel to identify fractions that appear to contain nearly pure prototype foamy virus integrase. Combine these fractions, and make a note of the volume while pipetting. This is typically eight to 10 milliliters.Using a spectrophotometer, measure the UV absorbance at 280 nanometers of the combined fractions, and calculate the total amount of prototype foamy virus integrase protein. A typical yield is around 10 milligrams per liter of induced culture. To remove the hexahistidine tag, supplement the prototype foamy virus integrase with a final concentration of 10-millimolar DTT and 0.1-millimolar EDTA.Then add 15 micrograms of human rhinovirus 3C protease per milligram of prototype foamy virus integrase, and incubate at four degrees Celsius overnight. To perform heparin affinity chromatography fractionation of the prototype foamy virus integrase, reduce the sodium chloride concentration of the sample to 200 millimolar by adding 1.5 volumes of buffer C.Then load the diluted prototype foamy virus integrase sample to the heparin Sepharose column at 0.5 milliliters per minute flow rate with a maximum pressure limit of one megapascal. After washing the column and collecting the gradient and final wash, analyze load, flowthrough, wash, and fractions by 8%SDS-PAGE.Then evaluate fractions by staining the gel with Coomassie blue. Prototype foamy virus integrase and the contaminating nuclease have different affinities for heparin Sepharose chromatography. Identify heparin Sepharose fractions that appear to contain nearly pure prototype foamy virus integrase to use in the nuclease assay.To begin the nuclease assay, combine three microliters of heparin fraction and 50 nanograms of three-kilobase supercoiled plasmid DNA and reaction buffer to a final volume of 15 microliters. Incubate at 37 degrees Celsius for 90 minutes. Stop the reaction with one milligram per milliliter Proteinase K and 0.5%SDS, and incubate at 37 degrees Celsius for one hour.Then add 3.5 microliters of 6X loading dye to the nuclease assay reactions, and load the entire volume on a 1%agarose gel. Run the gel at 100 volts at ambient temperature for one hour or until the dye front reaches the end of the gel. When electrophoresis is complete, a fluorescent scanner image of the gel should show that linear DNA runs true to size at three kb, supercoiled DNA runs faster to around two kb, and that relaxed circle DNA runs slower at close to 3.5 kb.Using image analysis software, calculate the pixel volume of the supercoiled, linear, and relaxed circle plasmid in each lane. Call the sum of the pixel volumes for these three DNA forms the total DNA value, and use it to calculate the percentage of linear and relaxed circles. Fractions that contain contaminating nuclease display a higher percentage of linear and relaxed circles compared to the negative control.Combine the heparin Sepharose fractions that do not display contaminating nuclease activity, and transfer to 10 millimeter, six to eight kilodalton molecular weight cut-off dialysis tubing. Dialyze at four degrees Celsius overnight against one liter of dialysis solution. The next day, recover the nuclease-free prototype foamy virus integrase sample from the dialysis tubing.Measure the absorbance at 280 nanometers, and calculate the final protein concentration. Aliquot the protein, and snap freeze in liquid nitrogen. Then store the aliquots at minus 80 degrees Celsius.The nuclease assay was performed as outlined in this video. Negative controls include plasmid DNA with no protein and a previous purification of wild-type prototype foamy virus integrase known to be free of nuclease. A positive control for contaminating nuclease activity is a previous purification of catalytically inactive prototype foamy virus integrase that is known to have contaminating nuclease.Following this procedure, other methods, like biochemical assays or single molecule imaging, can be performed in order to answer additional questions, like enzyme kinetics and dynamics. After watching this video, you should have a good understanding of how to identify and remove contaminating nuclease activity from a recombinant protein.

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