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 &#956;L of room temperature super optimal broth with catabolite repression (SOC) media. Incubate for 1 h at 37 &#730;C with 225 revolutions per minute (rpm) shaking.</li>
		<li>Plate 25 &#956;L of the mixture on a 100 mm x 15 mm Petri dish containing 25 mL of Luria broth (LB) agar (1 L LB agar: 10 g bacto-tryptone, 10 g NaCl, 5 g yeast extract, 15 g agar) and 100 &#956;g/mL ampicillin (100 mg/mL stock solution).</li>
		<li>Plate the remaining 75 &#956;L of the transformed cells on a second identical plate. Incubate the plates with the lids down overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Retrieve the plates of transformed E. coli. Use a single colony to inoculate 3 mL of LB (1 L LB: 10 g of bacto-tryptone, 10 g of NaCl, 5 g of yeast extract) supplemented with 100 &#956;g/mL ampicillin in a 14-mL round bottom polypropylene culture tube. Incubate for ~8 h at 37 &#730;C with 225 rpm shaking.
	<ol>
		<li>Add the 3 mL of culture to 50 mL LB supplemented with 100 &#956;g/mL ampicillin and 34 &#956;g/mL chloramphenicol (34 mg/mL stock solution) in a 250 mL Erlenmeyer flask. Incubate the culture overnight at 37 &#176;C with 225 rpm shaking.</li>
		<li>Transfer the 53 mL of culture to 1 L LB, 100 &#956;g/mL ampicillin, and 34 &#956;g/mL chloramphenicol in a 4 L Erlenmeyer flask. Incubate at 37 &#176;C with 225 rpm shaking.</li>
		<li>Measure the optical density at 600 nm (OD600) of the culture periodically. Initially, check the culture OD600 every h. As the culture reaches the desired OD600, check every 15 min to not overgrow. Grow the culture to an OD600 between 0.9 and 1.0. Transfer the flask to an ice bath and swirl to reduce the temperature of the culture.</li>
	</ol>
	</li>
	<li>Transfer 1 mL of the culture to a 1.5 mL tube for later analysis by denaturing SDS-PAGE analysis.
	<ol>
		<li>Pellet the cells in the 1.5 mL tube by centrifugation for 1 min in a microfuge at 14,000 x g at room temperature. Discard the supernatant. Resuspend the pellet in 150 &#956;L phosphate buffered saline (PBS) by pipetting. The PBS may be either with or without CaCl2 and MgCl2.</li>
		<li>Add 150 &#956;L of 2x SDS-PAGE sample buffer (150 mM Tris-HCl, pH 6.8, 1.2% SDS, 30% glycerol, 15% &#946;-mercaptoethanol (&#946;ME), 0.0018% bromophenol blue) and mix thoroughly by pipet or vortex. Boil for 3 min. Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Add to the bacterial culture: a final concentration of 0.25 mM isopropyl-beta-D-thiogalactopyranoside (IPTG, 0.25 M stock solution) and 50 &#956;M ZnCl2 (50 mM stock solution). IPTG induces expression of the PFV IN gene from the T7 promoter and ZnCl2 is added as a supplement for the correct folding of an amino terminal zinc finger domain in PFV IN. Incubate the culture at 25 &#176;C with shaking at 225 rpm for 4 h. Immediately transfer the flask to an ice bath. Save 1 mL of culture for SDS-PAGE analysis following the same protocol as above in 1.3.</li>
	<li>Transfer the bacterial culture to appropriately sized centrifuge bottles. For example, a 1 L culture may be transferred to four sterile disposable 250 mL conical bottom polypropylene bottles. Spin the bottles in a refrigerated tabletop centrifuge at 3000 x g for 20 min at 4 &#730;C. Discard supernatants by pouring.
	<ol>
		<li>Resuspend all of the pellets from a 1 L bacterial culture in a total volume of 25 mL (6.25 mL per bottle) cold PBS (with or without CaCl2 and MgCl2) by pipetting. Combine and transfer the bacterial suspensions to one 50 mL conical tube. Spin in a refrigerated centrifuge at 3,000 x g for 20 min at 4 &#176;C. Discard the supernatant by pouring or pipetting.</li>
	</ol>
	</li>
	<li>Resuspend the bacterial pellet in 10 mL resuspension buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 500 &#956;M phenylmethylsulfanoxide (PMSF) protease inhibitor) by pipetting. Snap freeze the pellet by placing the tube in a Dewar flask with liquid nitrogen sufficient to submerge the 50 mL tube. Carefully remove the tube from liquid nitrogen and store at -80 &#176;C. 
	Note: Bacterial pellets may be stored for several months at -80 &#176;C. We have seen no loss of active protein following -80 &#176;C storage for two years.</li>
	<li>Analyze the pre-induction and post-induction samples by 8.3 cm wide, 7.3 cm high, 0.75 mm thick SDS-PAGE gels with 1.5 mL 6% polyacrylamide stacking layer (125 mM Tris-HCl, pH 6.8, 6% acrylamide, 0.1% SDS, 0.1% ammonium persulfate, 0.001% TEMED) and 3.5 mL 10% polyacrylamide resolving layer (375 mM Tris-HCl, pH 8.8, 10% acrylamide, 0.1% SDS, 0.1% ammonium persulfate, 0.001% TEMED)12.
	<ol>
		<li>After pouring the stacking gel, insert a 10 well comb. When the gel has polymerized, assemble the PAGE apparatus, and add sufficient SDS-PAGE running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS).</li>
		<li>Load 10 &#956;L of each sample and 4 &#956;L of prestained protein standards. Run at 16.5 V/cm until the dye front reaches the bottom of the gel, approximately 60 min.</li>
		<li>Disassemble the gel plates and transfer the gel to a plastic tub. Add Coomassie Brilliant Blue staining solution (0.1% Coomassie brilliant blue R-250, 40% methanol, 10% acetic acid) to generously cover the gel and stain for 20 min at ambient temperature.</li>
		<li>Remove the staining solution by pouring and replace with destain solution (40% methanol, 10% acetic acid) until bands are readily visible. Remove the destaining solution by pouring and replace with deionized water. PFV IN should be easily identified in the post-induction sample. PFV IN with a hexahistidine tag has a molecular weight of 47374 Da.</li>
		<li>If PFV IN is not visible, repeat the induction starting with a different colony from one of the transformation plates.</li>
	</ol>
	</li>
</ol>
2. Nickel Affinity Chromatography
<ol>
	<li>Thaw one bacterial pellet on ice. 
	Note: This will take significant time (approximately 2 - 3 h). When the pellet has thawed to a cell slurry, add an additional 500 &#956;M PMSF (100 mM stock solution).</li>
	<li>Keep the 50 mL tube of bacteria slurry on ice. Sonicate the bacteria at 30% amplitude for 30 s (1 s on, 1 s off) per pellet derived from 1 L culture using a sonicator with a 0.5 inch diameter bio horn with a 0.125 inch diameter tapered microtip, a frequency of 20 kHz and maximum power of 400 W.</li>
	<li>Transfer the cell sample to a cold ultracentrifuge tube. Use polycarbonate bottle assemblies (25 mm diameter, 89 mm height) compatible with a Type 60 Ti fixed angle rotor. Alternative tubes and rotors may be used if the same gravitation force is achieved. Spin 120,000 x g for 60 min at 4 &#176;C. There should be an obvious pellet. The supernatant may have a yellow color.</li>
	<li>Transfer the supernatant by pouring or pipetting to a cold 50 mL conical tube. Note the volume; this volume should be approximately the volume of resuspension buffer used prior to snap freezing. If the supernatant is viscous, it may be contaminated by bacterial DNA which could clog the chromatography column. In this case, repeat the ultracentrifugation to pellet the bacterial DNA.</li>
	<li>Prepare 500 mL Buffer A (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 500 &#956;M PMSF) and 500 mL Buffer B (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 500 &#956;M PMSF, 200 mM imidazole). 
	Note: In this protocol, keep the fast protein liquid chromatography (FPLC) system, all buffers and the sample at 4 &#176;C in a cold room.
	<ol>
		<li>Sterile filter Buffers A and B with 0.2 &#956;m disposable filter units. Depending on the FPLC system, wash pump A and pump B with Buffer A and Buffer B, respectively. Flow 90% Buffer A and 10% Buffer B through the system until the conductivity and UV readings stabilize. The maximum flow rate will depend on the instrument; use a flow rate of 5 mL/min with a maximum pressure limit of 1.0 MPa.</li>
	</ol>
	</li>
	<li>Prepare a 110 mm long, 5 mm diameter FPLC column with 1.5 mL nickel-charged resin (maximum binding capacity 50 mg/mL, maximum pressure 1 MPa). The column may be prepared the day before purification and stored at 4 &#730;C.</li>
	<li>Connect the column to the FPLC instrument. Equilibrate the column with 20 mL of 90% Buffer A and 10% Buffer B (20 mM imidazole) at a flow rate of 0.5 mL/min with a maximum pressure limit of 0.5 MPa. The instrument should be collecting real time data of conductivity and UV absorbance at 280 nm (A280). The conductivity and UV readings should stabilize. If these readings have not stabilized, continue to flow buffers through the column until they are stable.</li>
	<li>Load the protein sample (~10 mL per pellet) to the column at a flow rate of 0.15 mL/min with a maximum pressure limit of 0.5 MPa. The flow rate and column pressure remains the same throughout the purification. Collect the flow through in a 50 mL tube.
	<ol>
		<li>Wash the column with 20 mL 90% Buffer A and 10% Buffer B. Collect the wash in a 50 mL tube. Elute the proteins with a 20 mL linear gradient of 10% to 100% Buffer B (20 mM to 200 mM imidazole).</li>
		<li>Finally, wash the column with 5 mL 100% Buffer B (200 mM imidazole). Collect the gradient and final wash in 0.27 mL fractions.</li>
	</ol>
	</li>
	<li>Combine 15 &#956;L of 2x SDS-PAGE sample buffer with 15 &#956;L of the flow through, wash, and peak A280 fractions. Boil the samples for 3 min.
	<ol>
		<li>Prepare two 10% SDS-PAGE gels as described previously in step 1.6 except with 15 well combs. Load 10 &#956;L of each sample to the gels and 4 &#956;L of prestained protein standards. Run, stain, and analyze the gels as described in step 1.6.</li>
		<li>Combine fractions that appear to contain nearly pure PFV IN based on visual inspection. Measure the volume by pipetting, typically 8 - 10 mL.</li>
		<li>Using a spectrophotometer, measure the A280 of the combined fractions and calculate the total amount of PFV IN protein; our typical yield is ~10 mg per L of induced culture. The extinction coefficient of PFV IN with the hexahistidine tag is 59360 cm-1 M-1.</li>
	</ol>
	</li>
	<li>To remove the hexahistidine tag, supplement the PFV IN with 10 mM dithiothreitol (DTT, 1 M stock solution) and 0.1 mM EDTA, pH 8.0 (0.5 M stock solution). Add 15 &#956;g of human rhinovirus (HRV) 3C protease per mg PFV IN. Incubate at 4 &#730;C overnight. Cleavage reduces the PFV IN molecular weight from 47374 Da to 44394 Da and may be verified by 10% SDS-PAGE analysis.</li>
</ol>
3. Heparin affinity chromatography
<ol>
	<li>Prepare 250 mL Buffer C (50 mM Tris-HCl, pH 7.5, 10 mM DTT, 0.1 mM EDTA, 500 &#956;M PMSF) and 250 mL Buffer D (50 mM Tris-HCl, pH 7.5, 10 mM DTT, 0.1 mM EDTA, 500 &#956;M PMSF, 1 M NaCl). 
	Note: In this protocol, the FPLC system, buffers, and sample are kept in a cold room at 4 &#730;C.
	<ol>
		<li>Sterile filter Buffers C and D with 0.2 &#956;m disposable filter units. Depending on the FPLC system, wash pump A and pump B with Buffer C and Buffer D, respectively. Flow 80% Buffer C and 20% Buffer D through the system until the conductivity and UV readings stabilize. The maximum flow rate will depend on the instrument; we routinely use a flow rate of 5 mL/min with a maximum pressure limit of 1.0 MPa.</li>
	</ol>
	</li>
	<li>Prepare an 80 mm long, 5 mm diameter FPLC column with 1 mL of heparin sepharose resin (maximum binding capacity 2.0 mg of bovine antithrombin III per mL resin; maximum pressure 1.4 MPa). The column may be prepared the day before purification and stored at 4 &#176;C. Connect the column to the FPLC instrument.
	<ol>
		<li>Equilibrate the column with 20 mL of 80% Buffer C and 20% Buffer D (200 mM NaCl) at a flow rate of 0.5 mL/min with a maximum pressure limit of 1 MPa. The instrument should be collecting real time data of conductivity and UV absorbance at 280 nm (A280). The conductivity and UV readings should stabilize. If these readings have not stabilized, continue to flow buffers through the column until they are stable.</li>
	</ol>
	</li>
	<li>Reduce the NaCl concentration of the PFV IN sample to 200 mM by adding 1.5 volumes Buffer C. For example, add 15 mL of Buffer C to 10 mL PFV IN. Our total volume is typically ~25 mL.</li>
	<li>Load the diluted PFV IN sample to the heparin sepharose column at 0.5 mL/min flow rate with maximum pressure limit 1.0 MPa. The flow rate and column pressure remains the same throughout the purification. Collect the flow through in a 50 mL conical tube.
	<ol>
		<li>Wash the column with 20 mL of 80% Buffer C and 20% Buffer D (200 mM NaCl), collecting the wash in a 50 mL conical tube. Elute the column with a 30 mL linear gradient of 20% to 100% Buffer D (200 mM to 1 M NaCl).</li>
		<li>Wash the column with 5 mL of 100% Buffer D. Collect the gradient and final wash in 0.37 mL fractions.</li>
	</ol>
	</li>
	<li>Analyze load, flow through, wash, and fractions by 8% SDS-PAGE as described in 2.9. PFV IN following cleavage of the hexahistidine tag has a molecular weight of 44394 Da and the extinction coefficient remains 59360 cm-1 M-1 without the hexahistidine tag. Store all fractions at 4 &#730;C until the completion of a nuclease assay.</li>
</ol>
4. Nuclease assay
<ol>
	<li>Identify heparin sepharose fractions that appear to contain nearly pure PFV IN. Combine 3 &#956;L of heparin fraction and 50 ng of 3 kb supercoiled plasmid DNA in reaction buffer (10 mM HEPES, pH 7.5, 110 mM NaCl, 5 mM MgSO4, 4 &#956;M ZnCl2, 10 mM DTT) with a final volume of 15 &#956;L. Incubate at 37 &#730;C for 90 min. Include a negative control with no PFV IN.
	<ol>
		<li>Stop the reaction with 1 mg/mL proteinase K (20 mg/mL stock solution) and 0.5% SDS (10% stock solution). Incubate at 37 &#176;C for 1 h. Samples may be stored at -20 &#176;C for later analysis.</li>
	</ol>
	</li>
	<li>Prepare a 125 mL gel of 1% weight per volume (w/v) agarose in 1x&#160;TAE buffer (40 mM Tris-acetate, 1 mM EDTA) with 0.1 &#181;g/mL ethidium bromide (10 mg/mL stock solution)13. Melt the agarose solution and pour to a 15 cm x 10 cm gel casting tray. Insert a 15 well comb with 5 mm wide, 0.75 mm thick wells. Thin wells yield better band resolution compared to 1.5 mm thick wells. Allow the gel to solidify at ambient temperature.
	<ol>
		<li>Immerse the gel in 1x&#160;TAE with 0.1 &#181;g/mL ethidium bromide. Add 3.5 &#956;L of 6x loading dye (50% glycerol, 0.15% Orange G dye) to the nuclease assay reactions. Load the entire volume to the gel. Run the gel at constant voltage, 10 volts per cm (100 V) at ambient temperature for 1 h or until the Orange G dye front reaches the end of the gel.</li>
	</ol>
	</li>
	<li>Immediately image the agarose gel with a fluorescent scanner set to detect ethidium bromide. Linear DNA should run true to size at 3 kb, supercoiled DNA should run faster (~2 kb), and relaxed circle DNA should run slower (~3.5 kb).
	<ol>
		<li>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 &#34;total DNA&#34; value and use it to calculate the percentage of linear and relaxed circles. For example, the pixel volume of relaxed circles is divided by the total DNA pixel value in that lane, multiply this number by 100 to determine a percentage. Fractions that contain contaminating nuclease display a higher percentage of linear and relaxed circles compared to the negative control.</li>
	</ol>
	</li>
	<li>Combine heparin sepharose fractions that do not display contaminating nuclease activity. Dialyze in 10 mm 6 - 8 kDa molecular weight cutoff (MWCO) dialysis tubing at 4 &#176;C overnight against 1 L of 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 50 &#956;M ZnCl2, 10 mM DTT, 20% glycerol.</li>
	<li>Recover the nuclease-free PFV IN sample from dialysis tubing. Measure the A280 and calculate the final protein concentration. Aliquot and snap freeze in liquid nitrogen. Store aliquots at -80 &#176;C.</li>
</ol>

<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>

The authors have nothing to disclose.

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 affinity for heparin sepharose that is readily distinguishable from the bacterial nuclease contaminant1. The key to preparation of nuclease-free PFV IN is the careful quantitation of nuclease activity in each fraction following gradient elution from heparin sepharose. The gradient must be sufficiently shallow to allow the separation of PFV IN from the nuclease contaminant; a steep gradient would not be compatible with efficient separation. In addition, the plasmid DNA substrate should have a low percentage of relaxed circles to reduce the background of the nuclease assay. If the plasmid DNA substrate has a high background of relaxed circles, a new purification of plasmid DNA should be performed. The nuclease activity assay reveals the presence of the contaminating nuclease and fractions that should be discarded.
In some cases, size exclusion chromatography (SEC) may be an effective method for removal of a contaminating nuclease2. Previous groups have used SEC during purification of PFV IN4. SEC has a limited load volume and tends to reduce the final protein concentration. The major advantage to affinity and ion exchange chromatography is the ability to load a significantly greater volume than SEC.
Testing for nuclease activity may be adapted to different proteins that co-purify with a bacterial nuclease. Here we identify that PFV IN and a contaminating nuclease may be separated by heparin sepharose chromatography. We have previously shown that PFV IN has a similar profile to the nuclease with Mono-S cation and Mono-Q anion exchange chromatography making these resins unsuitable for this protein. Depending on the affinity characteristics of the desired protein in relation to the nuclease, this protocol may be used with affinity or ion exchange resins and gradient elution. The key to effective removal of nuclease contamination from a protein of interest is a difference in affinity for a resin. Each fraction must be assayed for nuclease activity. The chromatography resins of this protocol may not be directly applied to all proteins that co-purify with bacterial nucleases. However, the overall concept of using an affinity or ion exchange resin to separate the nuclease from a protein of interest and assaying fractions for nuclease activity may be broadly applicable.

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.

<table><tbody><tr><td>BL21/DE3 Rosetta E. coli</td><td>EMD Millipore</td><td>70956-4</td><td></td></tr><tr><td>LB broth</td><td>EMD biosciences</td><td>1.10285.0500</td><td></td></tr><tr><td>Ampicillin</td><td>Amresco</td><td>0339</td><td></td></tr><tr><td>Chloramphenicol&amp;nbsp;</td><td>Amresco</td><td>0230</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>IPTG&amp;nbsp;</td><td>Denville Scientific</td><td>CI8280</td><td></td></tr><tr><td>ZnCl2</td><td>Sigma-Aldrich</td><td>208086</td><td></td></tr><tr><td>Tris Ultra Pure</td><td>Gojira Fine Chemicals</td><td>UTS1003</td><td></td></tr><tr><td>NaCl</td><td>P212121</td><td>RP-S23020</td><td></td></tr><tr><td>PMSF&amp;nbsp;</td><td>Amresco</td><td>0754</td><td></td></tr><tr><td>Imidazole&amp;nbsp;</td><td>Sigma-Aldrich</td><td>I0250</td><td></td></tr><tr><td>DTT&amp;nbsp;</td><td>P212121</td><td>SV-DTT</td><td></td></tr><tr><td>UltraPure EDTA</td><td>Invitrogen/Gibco</td><td>15575</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Amresco</td><td>0662</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Denville Scientific</td><td>CA3510</td><td></td></tr><tr><td>Ethidium bromide</td><td>Thermo Fisher Scientific</td><td>BP1302</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G37-20</td><td></td></tr><tr><td>Ni-NTA Superflow</td><td>Qiagen</td><td>30430</td><td></td></tr><tr><td>Heparin Sepharose 6 Fast Flow</td><td>GE Healthcare Life Sciences</td><td>17-0998-01</td><td></td></tr><tr><td>HRV14 3C protease&amp;nbsp;</td><td>EMD Chemicals</td><td>71493-3</td><td></td></tr></tbody></table>

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 (Figure 2). The fractions with nearly pure PFV IN are combined and the hexahistidine tag is cleaved by a protease. We have determined that PFV IN and the contaminating nuclease have different affinities for heparin sepharose chromatography (Figure 3)1. PFV IN fractions following heparin sepharose chromatography are incubated with a supercoiled plasmid to assay for relaxed circles and linearized plasmid, the products of nuclease activity (Figure 4). This assay allows the identification of protein fractions without the nuclease (Figure 5). These fractions may be combined, dialyzed, and frozen for future experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56605/56605fig1.jpg" /> 
Figure 1: Induction of PFV IN in E. coli . Cells from cultures before (lane 2) and after (lane 3) induction are analyzed for the presence of PFV IN, 47374 Da. Samples were separated by 10% SDS-PAGE and stained with Coomassie brilliant blue. Lane 1, molecular weight markers (kDa). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig1large.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/56605/56605fig2.jpg" /> 
Figure 2: Nickel affinity chromatography fractions. Soluble bacterial lysates were separated by nickel affinity chromatography. PFV IN with a hexahistidine tag was eluted with a linear gradient of 20 mM to 200 mM imidazole. Fractions were evaluated by 10% SDS-PAGE and stained with Coomassie brilliant blue. PFV IN, 47374 Da, is readily apparent in the sample loaded to the column (load), but is less apparent in the flow through or wash. Fractions that elute early in the imidazole gradient commonly display a slower mobility contaminant near 75 kDa as well as some faster mobility contaminants at or below 25 kDa. At higher concentrations of imidazole there are no readily apparent contaminants and PFV IN appears to be relatively pure. In this example, fractions #33 through #84 were combined and further purified. Molecular weight markers (kDa) are on the left of each gel. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56605/56605fig3.jpg" /> 
Figure 3: Heparin sepharose chromatography of PFV IN 1 . Following nickel affinity and protease removal of the hexahistidine tag, PFV IN (44394 Da) was fractionated by heparin sepharose. Proteins were eluted with a linear gradient from 200 mM to 1 M NaCl. Even fractions #12 to #44 were evaluated by 8% SDS-PAGE stained with Coomassie blue. Molecular weight markers (kDa) are on the left of each gel. These fractions were tested for nuclease activity. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56605/56605fig4.jpg" /> 
Figure 4: Nuclease assay of heparin sepharose fractions1 . Each heparin sepharose fraction was added to supercoiled plasmid DNA. Fraction numbers correlate to those shown in Figure 3. Following incubation, the nuclease reactions were deproteinated and separated by 1% agarose with ethidium bromide. Negative controls include plasmid DNA with no protein (Target Only) and a previous purification of wild type PFV IN known to be free of nuclease (PFV IN WT). A positive control for contaminating nuclease activity is a previous purification of catalytically inactive PFV IN that is known to have contaminating nuclease (PFV IN D128N). DNA size markers are shown in kb. Supercoiled plasmid (SC), linear plasmid (L), and relaxed circles (RC) are indicated. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56605/56605fig5.jpg" /> 
Figure 5: Quantitation of nuclease assays1. Agarose gels were scanned and pixel values were quantified for supercoiled (SC), linear (L), and relaxed circle (RC) bands in each lane. Fraction numbers correlate to those shown in Figures 3 and 4. The percentages of linear and relaxed circles were calculated using the pixel values and the equation shown. For example, the pixel values for the bands of fraction #12 are: 817636 supercoiled plasmid, 50467 linear plasmid, and 112052 relaxed circles. The total DNA pixel value for fraction #12 is the sum of these values, 980155. The percentage of linear DNA is 50467 multiplied by 100 for the percentage and divided by the total DNA value equaling 5.1%. The percentage of relaxed circles is 112052 multiplied by 100 and divided by the total DNA value equaling 11.4%. The sum of linear and relaxed circle percentages is 16.5%. Negative control plasmid DNA with no protein (No IN, bottom graph) indicates that this preparation of plasmid included 4.4% of the total DNA as linear or relaxed circles. A second negative control was a previous purification of wild type PFV IN (WT) that displayed 8.6% of the total DNA as linear or relaxed circles. These two control lanes indicate the background level of nicked or linearized plasmid present with the substrate alone and with PFV IN. A positive control is a previous purification of catalytically inactive PFV IN (D128N) which had 23% of the total DNA as linear and relaxed circles. The DNA exposed to heparin sepharose fractions #12 through #22 resulted in &#62;10% of the DNA as linear and relaxed circles. In this example, fractions #24 to #42 displayed &#60;10% conversion to linear and relaxed circles. These fractions were combined and dialyzed. Aliquots were frozen and stored at -80 &#176;C for future use. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56605/56605fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

detection-removal-nuclease-contamination-during-purification

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.0K Views.  The Ohio State University College of Medicine. Recombinante prototype schuimend virus integrase eiwit is het vaak besmet met een bacteriële nuclease tijdens het zuiveringsproces ongehinderd. Deze methode nuclease besmetting identificeert en verwijdert het uit de definitieve opstelling van het enzym.

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

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Biology

InSET: A Novel Tool for Unbiased Genome-Wide Functional Element Discovery

Identification of Functionally-Relevant Lentivirus Integration Sites in an Insertional Mutagenesis Cell Library

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens have made strides in exploring this, they often limit their scope to known genomic elements and may introduce non-specific effects. This underscores the urgent need for novel functional genomics tools that enable a deeper, unbiased understanding of genome functionality. This protocol introduces the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for identifying lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library. InSET facilitates the capture of genome-wide lentiviral integration sites, with next-generation sequencing used to detect and quantify flanking sequences. InSET's design enables the analysis of integration site abundance variations in phenotypic screens on a large scale, establishing it as a robust tool for forward genetics and for identifying functional genomic elements. A key benefit of InSET is its capacity to reveal previously unidentified genomic elements, including novel functional exons of both protein-coding and non-coding RNAs, independent of prior annotation. Overall, InSET holds significant value in studying the intricate complexity of the human genome and transcriptome, where many genomic elements await functional characterization.

Insertional mutagenesis is an essential tool in forward genetics for identifying functional genomic elements. Here, we describe the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for detecting lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library.

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens ...

Genetics

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

Bioengineering

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

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

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