Emulphogene

The LDs of recombinant yeast cultures resuspended in 10 mM potassium phosphate (pH 8) containing 10% glycerol (v/v) were treated separately with emulphogene (Sigma-Aldrich, USA) at final concentration of 0.2% (v/v) for 45 min. The mixture was then centrifuged at 100,000 × g for 45 min. The supernatant was taken and the concentration of solubilized proteins was estimated by Bradford assay and enzymatic activity. The solubilized fraction was used for protein purification. All steps were performed on ice (4°C). His-tagged PdCLO2 and PdCLO4 from in the supernatant were purified on a Ni-NTA Superflow column (Qiagen) under native (non-denaturing) conditions, according to the manufacturer's instructions. The purity of the recombinant proteins was confirmed by SDS-12% PAGE followed by Coomassie-Blue staining. For western blot experiment, proteins were fractionated by 12% SDS-PAGE and electroblotted to Immobilon-P membranes (Millipore Corp., Bedford, MA) using a mini-transblot transfer cell apparatus (Bio-Rad). For detection of the recombinant CLO:His fusion protein, a mouse monoclonal anti-His antibody and an anti-mouse antibody conjugated to peroxidase were used at 1:2000. Blots were developed using the ECL kit from Pierce.

The sensitivity of the cCFS to proteolytic activity was tested, employing a 2 h treatment with proteinase K at 37 °C at a final concentration of 10 mg/ml. After incubation, the cCFS was assayed for residual activity. In order to determine the effect on pH on bioactivity, the pH of the cCFS was adjusted to pH 2 using 1 M HCl and incubated at RT for 2 h, before neutralizing. The cCFS was subsequently assayed for residual activity. To determine the thermal stability, the cCFS was heated at 60 and 80 °C for 10, 30 or 60 min, respectively, after which each sample was assayed for residual activity. The cCFS was stored at -20 °C and 4 °C. At different time intervals, between 24 h and 120 days, the cCFS was assayed for residual activity. Containers used in the following experiments were coated with Emulphogene® (Polyoxyethylene 10 Tridecyl Ether, Sigma-Aldrich) in order to prevent binding of the active substance to the container surface.