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, 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.
Single molecule (SM) biophysics is a rapidly growing field changing the way we look at biological phenomena. This field has the unique ability to link fundamental laws of physics and chemistry to 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 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 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.
1. Induce PCD expression in E. coli
2. Nickel affinity chromatography purification of PCD
3. Nuclease activity assay
4. Size exclusion chromatography purification of PCD
5. PCA oxidation and nuclease activity assays
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 (Figure 2). PCD is eluted in 2 steps of imidazole concentrations. Chromatography fractions are analyzed by SDS-PAGE. Fractions of nearly pure PCD are concentrated and further purified by SEC (Figure 3). SEC fractions are individually analyzed for both PCA oxidation activity and nuclease activity (Figure 4). Fractions that displayed high oxidation activity and no apparent nuclease activity are assayed for protein concentration and kept in a -80 °C freezer for experimental use.
Figure 1: Induction of PCD in E. coli. (A) pVP91A-pcaHG is shown with the pcaG (α) and hexahistidine-tagged pcaH (β) PCD subunits. (B) Representative SDS-PAGE gel of PCD induction. Molecular weights are indicated on the left. The mobilities of 28.3 kDa hexahistidine-tagged pcaH and 22.4 kDa pcaG are on the right. Uninduced E. coli (Un), induced E. coli (In), the pellet following E. coli lysis and ultracentrifugation (P), the supernatant following ultracentrifugation to be loaded to a nickel column (S), representative fraction following nickel chromatography (Ni), and representative fraction following SEC (SE). This figure has been modified from a previous publication12. Please click here to view a larger version of this figure.
Figure 2: Nickel affinity chromatography purification of PCD. (A) Chromatogram of nickel affinity chromatography of PCD. The A280 is shown in blue and the percent concentration of Ni Buffer B is shown in red. The sample was loaded in a low 20 mM imidazole concentration. The flowthrough (Flw Thr) shows the soluble bacterial proteins that did not bind to the nickel resin. The column was washed with 20 mL of 20 mM imidazole buffer. A second 15 mL wash was performed with 125 mM imidazole. Elution of PCD was performed with 250 mM imidazole. Some PCD eluted in the presence of 125 mM imidazole, but the majority of the protein eluted in 250 mM imidazole. (B) Representative SDS-PAGE analysis of nickel affinity fractions. The load, flowthrough (Flw Thr), and first wash showed the successful induction of PCD, the soluble bacterial proteins that did not bind the nickel resin, and the minimal proteins observed during the first wash, respectively. Several fractions throughout the second wash and elution steps are shown. Fractions from the second wash included PCD protein but also displayed detectable higher molecular weight contaminants. Fractions from the elution step appeared to be free of contaminants. Molecular weights are shown on the left. Mobilities of pcaH and pcaG are shown on the right. (C) Agarose gel of nuclease assay. The nickel affinity column load, flowthrough, wash, and multiple fractions were tested for nuclease activity. A negative control (control) is the plasmid without added protein. A positive control (PCDa) is a commercially available PCD known to be contaminated with a DNA nuclease. DNA species are indicated on the right as small fragments (SF), supercoiled (SC), linear (LN), nicked circle (NC), and nicked dimer (ND). (D) Quantitation of the various DNA species observed in the agarose gel nuclease assay. The total pixel volume of each lane was measured. The pixel volume of each DNA species was determined and expressed as a percentage of the total pixel volume in the lane. The negative control was 81.7% supercoiled with 14.4% nicked circles. The positive control displayed a significant increase of 46.0% nicked circles. The load and flowthrough contained bacterial nucleases that converted the plasmid and contaminating bacteria DNA to small fragments. The first wash at 20 mM imidazole also appeared to contain significant nuclease activity, resulting in linear and nicked circles. Fractions 4-7 from the second wash at 125 mM imidazole also displayed significant nuclease activity (particularly, fractions 4 and 5 that generated observed linearized plasmid). Fractions 29-38 from the elution step appeared more similar to the negative control. In this example, fractions 29-38 were chosen to be combined, concentrated, and further purified by SEC. This figure has been modified from a previous publication12. Please click here to view a larger version of this figure.
Figure 3: SEC purification of PCD. (A) Chromatogram of SEC of PCD fractions following nickel affinity chromatography. The A280 is shown in blue and elution fractions are indicated. PCD eluted from SEC as a single apparent peak. (B) Representative SDS-PAGE analysis of SEC fractions 33-48. The load is the concentrated PCD following nickel affinity purification. Fractions 33-48 span the apparent SEC peak. No detectable contaminants were observed. (C) Agarose gel of nuclease assay. The SEC load and multiple fractions were tested for nuclease activity. A negative control (control) is the plasmid without added protein. A positive control (PCDa) is a commercially available PCD known to be contaminated with a DNA nuclease. DNA species are indicated on the left as supercoiled (SC), nicked circle (NC), and nicked dimer (ND). (D) Quantitation of the various DNA species observed in the agarose gel nuclease assay. The total pixel volume of each lane was measured. The pixel volume of each DNA species was determined and expressed as a percentage of the total pixel volume in the lane. The negative control was 82.1% supercoiled with only 13.7% nicked circles. The positive control displayed a significant increase of 64.8% nicked circles. The SEC load displayed no apparent nuclease activity due to judicious choice of fractions from the nickel affinity purification. Similarly, fractions 33-48 appeared similar to the negative control. For example, fraction 36 was 82.5% supercoiled and 13.2% nicked circle. In this example, fractions 36 and 37 were chosen to be quantified, frozen, and kept in a -80 °C freezer for future experimental use. This figure has been modified from a previous publication12. Please click here to view a larger version of this figure.
Figure 4: PCA oxidation and nuclease activity of PCD SEC fractions. PCA oxidation was measured by A290. As PCD oxidized the PCA molecule, the A290 decreased. PCA oxidation was measured every 20 s for 1 h. A negative control with no added PCD fraction (blue line) showed no change in A290, indicating the PCA molecule was stable. Data from three representative SEC fractions (36 in red, 33 in orange, 39 in yellow) show that purified PCD reduced the A290, indicating oxidation of PCA. This figure has been modified from a previous publication12. Please click here to view a larger version of this figure.
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.
Commercially available OSSs, including GODCAT and PCD, have been shown to include significant nuclease contamination11. It is possible to purchase PCD and employ SEC to remove the nuclease contaminant11. However, the price of commercially available PCD from one vendor increased 5 fold following the publication of that method. This method generates a highly active, nuclease free PCD heterodimer and can conceivably be performed within 1 week. In our experience, the amount of PCD generated by a 1 L culture (1-2 mg) is sufficient for one year of experiments (3 μg/experiment) in a productive laboratory with 2 fluorescent imaging systems.
Induction efficiency is key to the success of this method. If the PCD heterodimer is not efficiently induced and apparent by SDS-PAGE, the purification will be unsuccessful. Two alternative strategies may be tried. First, attempt induction from a different colony on the E. coli transformation plate. Second, we have had previous success with BL21, but an alternative E. coli strain for expression, such as BL21 pLysS, may be tried. Success of the PCA oxidation assay relies on minimal exposure of the substrate to the PCD before starting the assay. It is highly recommended to assemble the 96 well plate reactions on ice in a cold room and add the protein sample immediately before loading the plate to the reader.
Nickel affinity chromatography may be sufficient to identify fractions of pure PCD with no contaminating nuclease activity. In this case, it is possible to eliminate the SEC purification. However, the nickel chromatography fractions should be combined and dialyzed overnight at 4 °C in SEC running buffer. The glycerol present in the SEC running buffer is important for storage at -80 °C.
The authors have nothing to disclose.
This work was supported by NIH GM121284 and AI126742 to KEY.
2-Mercaptoethanol | Sigma-Aldrich | M3148 | βME |
30% acrylamide and bis-acrylamide solution, 29:1 | Bio-Rad | 161-0156 | |
Acetic acid, Glacial Certified ACS | Fisherl Chemical | A38C-212 | |
Agar, Granulated | BD Biosciences | DF0145-17-0 | |
AKTA FPLC System | GE Healthcare Life Sciences | AKTA Purifier: Box-900, pH/C-900, UV-900, P-900, and Frac-920 | |
Amicon Ultra-2 Centrifugal Filter Unit | EMD Millipore | UFC201024 | 10 kDa MWCO |
Ammonium iron(II) sulfate hexahydrate | Sigma | F-2262 | |
Ammonium Persulfate (APS) Tablets | Amresco | K833-100TABS | |
Ampicillin | Amresco | 0339-25G | |
Bacto Tryptone | BD Biosciences | DF0123173 | |
BD Bacto Dehydrated Culture Media Additive: Bottle Yeast Extract | VWR | 90004-092 | |
BIS-TRIS propane,>=99.0% (titration) | Sigma-Aldrich | B6755-500G | |
Bromophenol Blue | Sigma-Aldrich | B0126-25G | |
Coomassie Brilliant Blue | Amresco | 0472-50G | |
Costar 96–Well Flat–Bottom EIA Plate | Bio-Rad | 2240096EDU | |
DTT | P212121 | SV-DTT | |
Dulbecco's Phosphate Buffered Saline 500ML | Sigma-Aldrich | D8537-500ML | PBS |
Ethidium bromide | Thermo Fisher Scientific | BP1302 | |
Glycerol | Fisher Scientific | G37-20 | |
Granulated LB Broth Miller | EMD Biosciences | 1.10285.0500 | |
Hi-Res Standard Agarose | AGTC Bioproducts | AG500D1 | |
Imidazole | Sigma-Aldrich | I0250-250G | |
IPTG | Goldbio | I2481C25 | |
Leupeptin | Roche | 11017128001 | |
Lysozyme from Chicken Egg White | Sigma-Aldrich | L6876-1G | |
Magnesium Chloride Hexahydrate | Amresco | 0288-1KG | |
Microvolume Spectrophotometer, with cuvet capability | Thermo Fisher | ND-2000C | |
NaCl | P212121 | RP-S23020 | |
Ni-NTA Superflow (100 ml) | Qiagen | 30430 | |
Novagen BL21 Competent Cells | EMD Millipore | 69-449-3 | SOC media included |
Orange G | Fisher Scientific | 0-267 | |
Pepstatin | Gold Biotechnology | P-020-25 | |
PMSF | Amresco | 0754-25G | |
Protocatechuic acid | Fisher Scientific | ICN15642110 | PCA |
Sodium dodecyl sulfate | P212121 | CI-00270-1KG | |
SpectraMax M2 Microplate Reader | Molecular Devises | ||
Sterile Disposable Filter Units with PES Membrane > 250mL | Thermo Fisher Scientific | 09-741-04 | |
Sterile Disposable Filter Units with PES Membrane > 500mL | Thermo Fisher Scientific | 09-741-02 | |
Superose 12 10/300 GL | GE Healthcare Life Sciences | 17517301 | |
TEMED | Amresco | 0761-25ML | |
Tris Ultra Pure | Gojira Fine Chemicals | UTS1003 | |
Typhoon 9410 variable mode fluorescent imager | GE Healthcare Life Sciences | ||
UltraPure EDTA | Invitrogen/Gibco | 15575 | |
ZnCl2 | Sigma-Aldrich | 208086 |