Production of Yvrk-recombinant E. coli
The Yvrk gene-recombinant E. coli was produced as described previously[11]. B. subtilis strain 128 obtained from the Korean Gene Bank was cultured for 24 hours in Luria-Bertani (LB) broth (Difco, USA) in an aerobic environment of 95% O2/5% CO2 and temperature of 37°C. Cells were recovered by centrifugation at 5,000 × g at 4°C for 15 minutes. DNA was extracted from the cell pellet using a QIAamp DNA mini kit (Qiagen, USA).
The open reading frames and associated ribosome binding sites of B. subtilis 168 YvrK (GenBank™ accession no. 2832786) were amplified by polymerase chain reaction (PCR) using genomic DNA with the following oligonucleotides (shown 5′-3′): Yvrk-F, ATGAAAAAAC AAAATGACAT TCCG and Yvrk-R, TTTACTG CATTTCTTTT TCACTAC. The PCR products containing YvrK were digested with NcoI and SalI. The optimal reaction profile proved to be 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds for denaturation, 58°C for 30 seconds and 72°C for 30 seconds for annealing, and 72°C for 5 minute for primer extension. The PCR products were separated by gel electrophoresis in 2% agarose containing ethidium bromide, illuminated with ultraviolet light, and photographed for documentation.
The cloned Yvrk DNA segment was inserted into a pBAD/gIII-A vector containing a histidine tag. The DNA sequencing of the Yvrk-recombinant vector was done using an automated DNA sequencer (ABI Prism®, USA). The recombinant vector was transfected to non-pathogenic TOP 10 E. coli with a heat shock method involving 42°C for 90 seconds followed by 4°C for 10 minutes. The recombinant E. coli were cultured and selected in LB agar including ampicillin (100 μg/ml) at 37°C and 95% O2/5% CO2 for 24 hours.
pBy was grown at 37°C until an OD600 value of 0.5 with shaking. After heat-shocking at 42°C for 2 minutes[12], 5 μl/ml of serial dilution of L-arabinose (Invitrogen, USA; 0.02%, 0.2%, 2%, 20%) and 5 mM MnCl2 (Sigma-Aldrich, USA) were added. The cells were incubated at 37°C for 6 hours with shaking and harvested by centrifugation (13000 rpm, 10 minutes, 4°C) and stored at -80°C. Cells containing the empty vector were compared as negative control. The cell pellet was mixed with sodium dodecyl sulfate (SDS) sample buffer and visualized by 0.1% Brilliant Blue R (Sigma-Aldrich, USA) staining after separating by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Purification and characterization of recombinant enzyme
pBy cells were grown at 37°C until an OD600 value of 0.3 with shaking. After heat-shocking at 42°C for 2 minutes, 5 μl/ml of 2% L-arabinose and 5 mM MnCl2 were added. To increase the solubility of OXDC, the cells were incubated at 28°C for 24 hours and centrifuged. The cell pellet was stored at -80°C.
For purification under native conditions the cells pellet was thawed for 15 minutes and suspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Cells were incubated on ice for 30 minutes, after which lysozyme was added to 1 mg/ml. The solution was sonicated on ice and the cell lysate was centrifuged at 10,000 × g for 30 min at 4°C. Ni-NTA Superflow (Qiagen, USA) was added to cleared lysate and mixed gently by shaking (200 rpm on a rotary shaker) at 4°C for 60 minutes. The lysate-Ni-NTA mixture was loaded into a column (BioRad, USA) and washed twice with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), and the protein was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The cell lysate, wash and elutes were mixed with SDS sample buffer and evaluated by SDS-PAGE.
To determine the optimal pH and temperature, purified OXDC or PBS in pH 5, pH 6, or pH 7 was incubated with 5 mM MnCl2 and 1.5 mM sodium oxalate for 24 hours at 10°C, 20°C, 28°C, 37°C, 42°C, 60°C, or 70°C in atmospheric O2. The oxalate-degrading activity of purified OXDC protein was measured using an oxalate kit (Trinity Biotech, USA).
In vivo oxalate-degrading activity
This study was approved by the Institutional Animal Care and Use Committee at Seoul National University Hospital Biomedical Research Institute. A transient hyperoxaluric rat model was established using 8-week-old Sprague–Dawley male rats[13]. Rats were anesthetized with an intramuscular injection of an 8:2 mixture of ketamine (Yuhan, Korea) and rompun (Bayer, Germany). A midline laparotomy was performed and a p10 tube was inserted into the bladder. Urine was collected hourly through the p10 tube before and until 3 hours after oral oxalate intake together with homogenates of pBy and TOP 10 E. coli (control). For oral oxalate intake, 0.5 ml of 1 mM sodium oxalate solution and 0.5 ml of pBy homogenates was administered through gastric gavage. Oxalate concentration in the hourly-collected urine was measured by a commercial oxalate kit.