This work was supported by NIH GM121284 and AI126742 to KEY.

1. Induce PCD expression in E. coli
<ol>
	<li>Combine 1 &#956;L pVP91A-pcaHG PCD expression plasmid (20 ng/&#956;L, Figure 1A) and 20 &#956;L of E.coli BL21 (20 &#956;L commercially available cells, &#62; 2 x 106 cfu/&#956;g plasmid) in a tube. Flick the tube to mix. Place the tube on ice 5 min.</li>
	<li>Place transformation at 42 &#176;C for 30 s. Then ice 2 min.</li>
	<li>Add 80 &#956;L SOC media (super optimal broth with catabolite repression: 2.5 mM KCl, 10 mM NaCl, 2% tryptone, 0.5% yeast extract, 10 mM MgSO4, 10 mM MgCl2, 20 mM glucose). Shake at 225 revolutions per min (rpm) 37 &#176;C for 1 h.</li>
	<li>Plate the transformation reaction on LB Amp Agar (1L Luria Broth agar: 10 g NaCl, 10 g bacto-tryptone, 5 g yeast extract, 15 g agar, 50 &#956;g/mL ampicillin; 25 mL per 10 cm diameter petri dish).</li>
	<li>Incubate the plate, lid facing down, at 37 &#176;C for 16-18 h.</li>
	<li>Inoculate 50 mL LB Amp (1L LB: 10 g bacto-tryptone, 10 g NaCl, 5 g yeast extract, 50 &#956;g/mL ampicillin) in a 250 mL Erlenmeyer flask with one colony. Incubate at 37 &#176;C for 16-18 h shaking at 225 rpm.</li>
	<li>Transfer 20 mL culture to a 4 L flask with 1 L LB Amp. Shake at 225 rpm at 37 &#176;C.</li>
	<li>Every h measure the culture OD600 (optical density at 600 nm). As the culture OD600 nears 0.5, increase the frequency of measurements to every 15 min. The desired density of the culture is 0.5 OD600.</li>
	<li>Transfer the 4L flask to a bin of ice. Swirl the flask in the ice bath to reduce the culture temperature. 
	Note: The time on ice should be kept to a minimum so that the cells remain metabolically active. Ideally the cells will be on ice less than 10 min.</li>
	<li>Successful induction of PCD can be observed by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Harvest 1 mL of the uninduced culture in a tube. Spin the sample 1 min at 14,000 x g in a microfuge at ambient temperature. Decant the supernatant.
	<ol>
		<li>Solubilize the pelleted cells in 150 &#956;L PBS (phosphate buffered saline).</li>
		<li>Add an equal volume of 2X loading dye (1.2% SDS, 30% glycerol, 150 mM Tris-HCl, pH 6.8, 0.0018% bromophenol blue, 15% &#946;-mercaptoethanol). Vortex the sample to mix thoroughly.</li>
		<li>Boil the sample for 3 min and transfer to ice. The sample can be stored in a -20 &#176;C freezer for future analysis. 
		Note: PBS is commercially available with or without CaCl2 and MgCl2. Many laboratories will have PBS without CaCl2 and MgCl2 for cell culture methods. We have found no difference with PBS with or without CaCl2 and MgCl2.</li>
	</ol>
	</li>
	<li>Transfer the 4 L flask to an incubator at 17 &#176;C with 180 rpm shaking. Continue to monitor the OD600 every 20 min.</li>
	<li>At 0.7 OD600 add isopropyl-beta-D-thiogalactopyranoside (IPTG) to 0.5 mM final concentration (0.25 M stock solution) and 10 mg/L ammonium iron (II) sulfate hexahydrate (Fe(NH4)2(SO4)2, 10 mg/mL stock solution). PCD genes pcaH and pcaG are induced from the T5 promoter by the addition of IPTG (Figure 1A). Iron sulfate is bound by PCD and required for catalytic activity by coordinating oxygen during catechol opening.</li>
	<li>Shake the culture at 180 rpm at 17 &#176;C for 18 h.</li>
	<li>Place the culture flask in ice as in 1.2. Harvest 1 mL of the induced cells for SDS-PAGE as in 1.3.</li>
	<li>Pour the bacterial culture to bottles appropriate for centrifugation. A 1 L culture may be centrifuged in 4 250 mL conical bottom bottles. Pellet the culture at 4 &#176;C for 20 min at 3000 x g. Decant the supernatants. Dispose of bacterial liquid waste appropriately.</li>
	<li>Pipet to resuspend the pellets in 25 mL cold PBS (CaCl2 and MgCl2 are optional) per 1 L culture.</li>
	<li>Transfer the resuspension to 50 mL conical tubes (1 50 mL tube per 1 L culture). Pellet the cells at 3000 x g for 20 min at 4 &#176;C. Decant the supernatant and dispose appropriately.</li>
	<li>Resuspend the cells in 10 mL of lysis buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 20 mM imidazole, 10 % glycerol, 800 ng/mL Pepstatin, 1 &#956;g/mL Leupeptin, and 87.1 &#956;g/mL phenylmethylsulfonyl fluoride (PMSF)) by pipetting. Freeze the resuspension with liquid nitrogen in a Dewar flask. Store the sample tubes in a -80 &#176;C freezer. We have purified PCD from pellets stored at -80 &#176;C for one year with no apparent loss of activity.</li>
	<li>Compare the uninduced and induced cells by SDS-PAGE. 
	Note: We have had no difficulty with induction of PCD heterodimer. However, we recommend testing the induction before continuing with the purification in the event any reagent has expired unexpectedly.
	<ol>
		<li>Assemble plates for SDS-PAGE (dimensions: 7.3 cm x 8.3 cm x 0.75 mm thick). The stacking gel is 1.5 mL 6% polyacrylamide (6% acrylamide, 125 mM Tris-HCl, pH 6.8, 0.1% SDS, 0.1% ammonium persulfate, 0.001% TEMED). The resolving gel is 3.5 mL 12% polyacrylamide (12% acrylamide, 375 mM Tris-HCl, pH 8.8, 0.1% SDS, 0.1% ammonium persulfate, 0.001% TEMED). Insert a 10 well comb to the stacking layer.</li>
		<li>Load 10 &#956;L of uninduced and induced bacterial samples. Load 4 &#956;L of prestained molecular weight markers for proteins (Figure 1B).</li>
		<li>Electrophorese the gel at 16.5 V/cm approximately 1 h. The bromophenol blue should reach the bottom of the gel.</li>
		<li>Place the gel in Coomassie Blue stain (10% acetic acid, 40% methanol, 0.1% Coomassie Blue dye) in a plastic tub. The stain should completely immerse the gel. Stain at ambient temperature for 20 min. Gentle rotation during staining is optional.</li>
		<li>Replace the solution with destain (10% acetic acid, 40% methanol). Incubate at ambient temperature with optional gentle rotation until protein bands are readily visible.</li>
		<li>Replace the destain with deionized water. Among the bacterial proteins, the induced PCD subunits should be visible in the induced cells. Hexahistidine tagged PCD subunit pcaH has a molecular weight of 28.3 kDa and the pcaG subunit is 22.4 kDa. If the PCD subunits are not apparent, a new induction derived from a novel colony should be performed.</li>
	</ol>
	</li>
</ol>
2. Nickel affinity chromatography purification of PCD
<ol>
	<li>Thaw on ice one 50 mL tube of induced cells. It may take 2-3 h to completely thaw the sample.</li>
	<li>Keeping the tube on ice, sonicate the sample at 30% amplitude for 1 min, cycling 1 s on and off. Use a tapered microtip (diameter 0.125 inches) sonicator. Maximum power is 400 W and frequency is 20 kHz and per 1 L culture pellet.</li>
	<li>Following sonication, add lysozyme to 0.2 mg/mL final concentration (10 mg/ml stock solution) and keep on ice for 30 min at 4 &#176;C.</li>
	<li>Pour the bacterial lysate into a pre-chilled polycarbonate bottle (dimensions: 25 mm x 89 mm). The bottle should be compatible with a fixed angle ultracentrifuge rotor. Other tubes and/or rotors may be substituted, but the final gravitation force should be maintained.</li>
	<li>Centrifuge for 60 min at 120,000 x g at 4 &#176;C. Cellular debris will form a pellet. The supernatant may appear yellow.
	<ol>
		<li>The pellet may be included in subsequent SDS-PAGE analysis to determine the solubility of PCD (Figure 1B). Solubilize the pellet by vortexing in 10 mL PBS. Transfer 150 &#956;L to a 1.5 mL tube. Prepare the sample for SDS-PAGE as in 1.3.</li>
	</ol>
	</li>
	<li>Pour the supernatant to a cold 50 mL conical tube. Note the volume. Contamination of the supernatant with bacterial DNA may yield a viscous sample. The bacterial genomic DNA could block the column flow. The ultracentrifugation step 2.2 should be repeated to pellet the bacterial DNA. A pellet from this second spin may not be readily visible or may be transparent.</li>
	<li>Make 500 mL Ni Buffer A (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 10% glycerol, 800 ng/mL Pepstatin, 1 &#956;g/mL Leupeptin, and 87.1 &#956;g/mL PMSF) and 500 mL Ni Buffer B (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10% glycerol, 800 ng/mL Pepstatin, 1 &#956;g/mL Leupeptin, and 87.1 &#956;g/mL PMSF, 250 mM imidazole, pH 8.0). Pass both Ni Buffers through 0.2 &#956;m pore filters.</li>
	<li>The sample, buffers, and the FPLC (fast protein liquid chromatography) system are in a refrigerated room at 4 &#176;C. Wash pump A with Ni Buffer A and pump B with Ni Buffer B. Wash the system with 20 mM imidazole (8% Ni Buffer B, 92% Ni Buffer A) until the UV and conductivity stabilize. We routinely flow buffers at 5 mL/min with a 1.0 MPa pressure limit. The flow rate and pressure limit should be determined by the specifications of the FPLC instrument used.</li>
	<li>Prepare a column with 1.5 mL nickel-charged resin (dimensions: 110 mm long x 5 mm). The resin binding capacity is 50 mg/mL and can tolerate 1 MPa pressure. A column may be poured and stored at 4 &#176;C before purification. 
	Note: The size of the column may be proportionally increased to accommodate more than one 50 mL tube of induced cells if more protein is required. We prefer a fresh column for each preparation to ensure that no residual proteins contaminate our desired protein. However, nickel resins may be recycled according to manufacturer&#39;s instructions.</li>
	<li>Attach the column of nickel-charged resin to the FPLC. Run 20 mL of 92% Ni Buffer A and 8% Ni Buffer B (20 mM imidazole) at 0.5 mL/min with a 0.5 MPa pressure limit through the column to equilibrate. In real time the FPLC should measure A280 (280 nm UV absorbance) as well as conductivity. If these values have not stabilized after 20 mL volume has passed through the column, flow the buffers until they have stabilized.</li>
	<li>Load the sample to the column (~10 mL) at 0.15 mL/min. Set the pressure limit to 0.5 MPa. Collect the flow through.</li>
	<li>Wash the column with 20 mL Ni Buffer at 20 mM imidazole (92% Ni Buffer A and 8% Ni Buffer B). Retain the wash in a 50 mL tube for analysis. Wash the column with 15 mL of 50% Ni Buffer A and 50% Ni Buffer B (125 mM imidazole). Collect the elution in 19 fractions of 0.8 mL volume. Wash the column with 15 mL 100% Ni Buffer B. Collect an additional 75 fractions of 0.2 mL. Some PCD heterodimer will elute in the 50% Ni Buffer B wash, but the majority of the heterodimer will elute in the 100% Ni Buffer B wash.</li>
	<li>Analyze collected fractions on 12% SDS-PAGE gels to confirm presence of PCD. Add equal volumes of 2X loading dye to the flow through, wash, and peak A280 fractions. Boil for 3 min. Transfer to ice. Pour two 12% SDS-PAGE gels (as in step 1.7). Repeat the gel method as described in 1.7.</li>
</ol>
3. Nuclease activity assay
<ol>
	<li>Based on the SDS-PAGE analysis, identify nickel affinity fractions that contain nearly pure PCD. Combine 5 &#956;L chromatography fraction and 500 ng 3 kb supercoiled plasmid pXba+ in reaction buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.1 mM DTT) with a final volume of 50 &#956;L.
	<ol>
		<li>Incubate at 37 &#176;C for 1 h.</li>
		<li>Include a negative control (no added protein) and a positive control (commercially available PCD). Stop the reaction with 10 &#956;L stop solution (150 mM EDTA, pH 8.0, 0.6% SDS, 18% glycerol, 0.15% Orange G).</li>
		<li>Samples may be kept in a -20 &#176;C freezer to be analyzed later. Any supercoiled plasmid may be used in a nuclease assay as long as the supercoiled and relaxed circle reaction products may be resolved by agarose gel electrophoresis.</li>
	</ol>
	</li>
	<li>Pour a 120 mL 1% agarose gel in 1X TAE ethidium buffer (40 mM Tris-acetate, 1 mM EDTA, 0.5 &#181;g/mL ethidium bromide) in a gel cast (dimensions: 15 x 10 cm). Use a 15 well comb (well dimensions: 5mm x 1.5 mm). When the gel has set, immerse it in 1X TAE ethidium buffer.</li>
	<li>Analyze 30 &#956;L of the reactions by agarose gel. Electrophorese at 10 V/cm at ambient temperature for approximately 1 h. The Orange G dye front should be at the end of the gel.</li>
	<li>Use a fluorescence scanner to immediately image the ethidium bromide signal of the gel. If a 3 kb plasmid was used, the slowest band will be relaxed circles at ~3.5 kb, linear DNA will run at 3 kb, and supercoiled plasmid will have the fastest mobility at ~2 kb.
	<ol>
		<li>Calculate the total pixel volume of each lane with image analysis software.</li>
		<li>Determine the pixel volume of the various DNA species, such as supercoiled, linear, and nicked circles. Use these values to determine the percentage of each DNA species. For example, increased presence of nicked circles associated with a fraction compared to the negative control indicates the presence of nuclease. The pixel volume of nicked circles in a lane is divided by the pixel value of total DNA. Determine a percentage by multiplying this number by 100.</li>
	</ol>
	</li>
	<li>Combine fractions from the second elution peak that contain nearly pure PCD heterodimer based on SDS-PAGE analysis and have minimal to undetectable nuclease activity. Our typical pooled volume is ~2 mL.</li>
	<li>Load the sample to a centrifugal filter unit with a 10 kDa molecular weight cutoff. Centrifuge in a swinging bucket centrifuge at 4000 x g for 40 min at 4 &#176;C. Alternatively a 35&#176; fixed angle rotor may be used at 7500 x g for 20 min at 4 &#176;C.
	<ol>
		<li>Repeat the centrifugation until the final retentate volume is 100-200 &#956;L.</li>
		<li>Invert the filter unit and recover the retentate by centrifugation at 1000 x g for 2 min at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Size exclusion chromatography purification of PCD
<ol>
	<li>Make 250 mL size exclusion chromatography (SEC) running buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10% glycerol, 0.1 mM EDTA, 800 ng/mL Pepstatin, 1 &#956;g/mL Leupeptin, and 87.1 &#956;g/mL PMSF). Pass the buffer through a 0.2 &#956;m pore filter and store at 4 &#176;C. 
	Note: Perform all steps in a refrigerated room at 4 &#176;C. SEC purification is optional, but the protein should be stored in SEC running buffer. If SEC purification is omitted, the retentate collected in 3.6.2. should be dialyzed against 1 L of SEC buffer in 10 kDa MWCO (molecular weight cut off) dialysis tubing at 4 &#176;C overnight.</li>
	<li>Equilibrate a cross-linked agarose SEC (size exclusion chromatography) column (dimesions: 10 mm x 300 mm; 24 mL bed volume; 25 - 500 &#956;L sample volume; 1.5 MPa pressure limit; 2 x 106 Da exclusion limit; 1 to 300 kDa separation) with SEC Running Buffer at 0.5 mL/min. 
	Note: If desired alternative size SEC columns may be used.</li>
	<li>Load the concentrated fractions to a 200 &#956;L volume injection loop. Load the sample at 0.5 mL/min to the column. SEC resolution increases with smaller load volume. Elute with 23 mL SEC running buffer and collect 94 fractions of 250 &#956;L. The SEC chromatogram should resolve a single A280 peak that is the PCD heterodimer. We find that PCD elutes 8.9 mL. The elution timing will change with alternative SEC columns.</li>
</ol>
5. PCA oxidation and nuclease activity assays
<ol>
	<li>Reactions to assay both oxidation of PCA and nuclease activity are performed in a 96-well flat-bottom plate. Assemble reactions in a 96 well plate on ice in a 4 &#176;C cold room to prevent premature catalysis. Combine in a final volume of 50 &#956;L: 130 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 0.1 mM DTT, 5 mM PCA, 10 ng/mL supercoiled plasmid pXba+, and 10 &#956;L of individual PCD SEC fractions. 
	Note: The PCD SEC fractions should be added last and immediately before analysis as the protein will begin catalysis at the time of addition.</li>
	<li>PCD oxidation of PCA results in reduced absorbance of PCA at 290 nm (A290). Transfer the 96 well plate to the plate holder of a plate reader set to an internal temperature of 37 &#176;C. Retract the plate holder into the instrument and measure A290 at 20 s intervals for 1 h. Have the instrument shake the plate 5 s before each reading.</li>
	<li>After 1 h, terminate the reactions by adding 10 &#956;L stop solution (150 mM EDTA, pH 8.0, 0.6% SDS, 18% glycerol, 0.15% Orange G).</li>
	<li>Prepare, load, and run an agarose gel as in step 3.2.</li>
	<li>Image and analyze the agarose gel as in step 3.3.</li>
	<li>Select fractions with the most PCA oxidation activity and no observed nuclease contamination for long-term storage at -80 &#176;C. Measure the A280.</li>
	<li>Calculate the total PCD concentration using the A280 and the extinction coefficient (&#949;280) of 734,700 M-1cm-1.</li>
	<li>Snap freeze individual fractions in liquid nitrogen. Store in a -80 &#176;C freezer. Alternatively, combine active, nuclease-free fractions, aliquot, and freeze in the same way. Our typical yield is 1-2 mg PCD per L culture. Typical use in a SM experiment is 3 &#956;g PCD. We have used PCD stored at -80 &#176;C for up to 1 year with no decrease in activity.</li>
</ol>

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The authors have nothing to disclose.

Oxygen scavenging systems are commonly included in single molecule fluorescence microscopy to reduce photobleaching3,7,8. These microscopy techniques are often used to observe nucleic acids or protein interactions with nucleic acids1,13,14. Contamination of OSSs with nucleases may lead to spurious results.
Co...

Single molecule (SM) biophysics is a rapidly growing field changing the way we look at&#160;biological phenomena. This field has the unique ability to link fundamental laws of physics and chemistry to&#160;biology. Fluorescence microscopy is one biophysical method that can achieve SM sensitivity. Fluorescence is used to detect biomolecules by linking them to small organic fluorophores or quantum dots1. These molecules can emit photons when excited by lasers before photobleaching irreversibly2. Photobleaching occurs when the fluorescent labels undergo chemical damage&#160;which destroys their ability to excite at the desired wavelength2,3. The presence of reactive oxygen species (ROS) in aqueous buffer are a primary cause of photobleaching2,4. Additionally, ROS can damage biomolecules and lead to erroneous observations in SM experiments5,6. To prevent oxidative damage, oxygen scavenging systems (OSS) can be used3,7,8. The glucose oxidase/catalase (GODCAT) system is efficient at removing oxygen8, but it produces potentially damaging peroxides as intermediates. These may be damaging to biomolecules of interest in SM studies.
Alternatively, protocatechuate 3,4 dioxygenase (PCD) will efficiently remove O2 from an aqueous solution using its substrate protocatechuic acid (PCA)7,9. PCD is a metalloenzyme that uses nonheme iron to coordinate PCA and catalyze the catechol ring opening reaction using dissolved O210. This one step reaction is shown to be an overall better OSS for improving fluorophore stability in SM experiments7. Unfortunately, many commercially available OSS enzymes, including PCD, contain contaminating nucleases11. These contaminants can lead to the damage of nucleic acid-based substrates used in SM experiments. This work will elucidate&#160;a chromatography-based purification protocol for the use of recombinant PCD in SM systems. PCD can be broadly applied to any experiment where ROS are damaging substrates needed for data acquisition.

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td>&amp;beta;ME</td></tr><tr><td>30% acrylamide and bis-acrylamide solution, 29:1</td><td>Bio-Rad</td><td>161-0156</td><td></td></tr><tr><td>Acetic acid, Glacial Certified ACS</td><td>Fisherl Chemical</td><td>A38C-212</td><td></td></tr><tr><td>Agar, Granulated</td><td>BD Biosciences</td><td>DF0145-17-0</td><td></td></tr><tr><td>AKTA FPLC System</td><td>GE Healthcare Life Sciences</td><td>AKTA Purifier: Box-900, pH/C-900, UV-900, P-900, and Frac-920</td><td></td></tr><tr><td>Amicon Ultra-2 Centrifugal Filter Unit</td><td>EMD Millipore</td><td>UFC201024</td><td>10 kDa MWCO</td></tr><tr><td>Ammonium iron(II) sulfate hexahydrate</td><td>Sigma</td><td>F-2262</td><td></td></tr><tr><td>Ammonium Persulfate (APS) Tablets</td><td>Amresco</td><td>K833-100TABS</td><td></td></tr><tr><td>Ampicillin</td><td>Amresco</td><td>0339-25G</td><td></td></tr><tr><td>Bacto Tryptone</td><td>BD Biosciences</td><td>DF0123173</td><td></td></tr><tr><td>BD Bacto Dehydrated Culture Media Additive: Bottle Yeast Extract</td><td>VWR</td><td>90004-092</td><td></td></tr><tr><td>BIS-TRIS propane,&gt;=99.0% (titration)</td><td>Sigma-Aldrich</td><td>B6755-500G</td><td></td></tr><tr><td>Bromophenol Blue</td><td>Sigma-Aldrich</td><td>B0126-25G</td><td></td></tr><tr><td>Coomassie Brilliant Blue</td><td>Amresco</td><td>0472-50G</td><td></td></tr><tr><td>Costar 96&amp;ndash;Well Flat&amp;ndash;Bottom EIA Plate</td><td>Bio-Rad</td><td>2240096EDU</td><td></td></tr><tr><td>DTT</td><td>P212121</td><td>SV-DTT</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline 500ML</td><td>Sigma-Aldrich</td><td>D8537-500ML</td><td>PBS</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>Granulated LB Broth Miller</td><td>EMD Biosciences</td><td>1.10285.0500</td><td></td></tr><tr><td>Hi-Res Standard Agarose</td><td>AGTC Bioproducts</td><td>AG500D1</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I0250-250G</td><td></td></tr><tr><td>IPTG</td><td>Goldbio</td><td>I2481C25</td><td></td></tr><tr><td>Leupeptin</td><td>Roche</td><td>11017128001</td><td></td></tr><tr><td>Lysozyme from Chicken Egg White</td><td>Sigma-Aldrich</td><td>L6876-1G</td><td></td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>Amresco</td><td>0288-1KG</td><td></td></tr><tr><td>Microvolume Spectrophotometer, with cuvet capability</td><td>Thermo Fisher</td><td>ND-2000C</td><td></td></tr><tr><td>NaCl</td><td>P212121</td><td>RP-S23020</td><td></td></tr><tr><td>Ni-NTA Superflow (100 ml)</td><td>Qiagen</td><td>30430</td><td></td></tr><tr><td>Novagen BL21 Competent Cells</td><td>EMD Millipore</td><td>69-449-3</td><td>SOC media included</td></tr><tr><td>Orange G</td><td>Fisher Scientific</td><td>0-267</td><td></td></tr><tr><td>Pepstatin</td><td>Gold Biotechnology</td><td>P-020-25</td><td></td></tr><tr><td>PMSF</td><td>Amresco</td><td>0754-25G</td><td></td></tr><tr><td>Protocatechuic acid</td><td>Fisher Scientific</td><td>ICN15642110</td><td>PCA</td></tr><tr><td>Sodium dodecyl sulfate</td><td>P212121</td><td>CI-00270-1KG</td><td></td></tr><tr><td>SpectraMax M2 Microplate Reader</td><td>Molecular Devises</td><td></td><td></td></tr><tr><td>Sterile Disposable Filter Units with PES Membrane &gt; 250mL</td><td>Thermo Fisher Scientific</td><td>09-741-04</td><td></td></tr><tr><td>Sterile Disposable Filter Units with PES Membrane &gt; 500mL</td><td>Thermo Fisher Scientific</td><td>09-741-02</td><td></td></tr><tr><td>Superose 12 10/300 GL</td><td>GE Healthcare Life Sciences</td><td>17517301</td><td></td></tr><tr><td>TEMED</td><td>Amresco</td><td>0761-25ML</td><td></td></tr><tr><td>Tris Ultra Pure</td><td>Gojira Fine Chemicals</td><td>UTS1003</td><td></td></tr><tr><td>Typhoon 9410 variable mode fluorescent imager</td><td>GE Healthcare Life Sciences</td><td></td><td></td></tr><tr><td>UltraPure EDTA</td><td>Invitrogen/Gibco</td><td>15575</td><td></td></tr><tr><td>ZnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>208086</td><td></td></tr></tbody></table>

expression and purification of nuclease-free oxygen scavenger protocatechuate 3,4-dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Commercially available oxygen scavenger PCD is frequently contaminated with a DNA nuclease. Contaminating nuclease activity could lead to spurious results in fluorescent studies, particularly studies that analyze DNA or DNA interacting proteins.We have found that recombinant PCD, a heterodimer of hexahistidine tagged pcaH and pcaG, may be expressed in E. coli (Figure 1). The heterodimer is first purified by nickel affinity chromatography (<strong class="xfig"...

Watch this Scientific Journal Video about Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase at JoVE.com

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

expression-purification-nuclease-free-oxygen-scavenger

Research

JoVE Journal

Biochemistry

6.3K Views.  The Ohio State University. Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

Video: Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Light-driven Enzymatic Decarboxylation

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation.

We describe a protocol for the light-catalyzed generation of hydrogen peroxide &#8212; a cofactor for oxidative transformations.

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. ...

Chemistry

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

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.

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 ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the ...

Genetics

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

Bioengineering

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or genome sequencing projects, are often difficult to provide for further studies or third parties due to the consumptive nature of the samples. Although now widely distributed over the Pacific coasts of Asia, Australia and North America, individual Pacific oyster specimens are genetically quite diverse and are therefore not directly suitable as the starting material for gene libraries. In this article, we demonstrate the use of unreferenced Pacific oyster specimens obtained from regional seafood markets to generate cDNA libraries. These libraries were then compared to the publicly available oyster genome, and the closest related library was selected using the mitochondrial reference genes Cytochrome C Oxidase subunit I (COX1) and NADH Dehydrogenase (ND). The suitability of the generated cDNA library is also demonstrated by cloning and expression of two genes encoding the enzymes UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS), which are responsible for the biosynthesis of UDP-xylose from UDP-glucose.

We describe a strategy for how to use RNA samples from unreferenced Pacific oyster specimens, and evaluate the genetic material by comparison with publicly available genome data to generate a virtually sequenced cDNA library.

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or ...

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Summary

Explore More Videos

Chapters in this video

Light-driven Enzymatic Decarboxylation

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

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

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters

Protease- and Acid-catalyzed Labeling Workflows Employing ¹⁸O-enriched Water