Protein methodology

2.5.1 TCA Precipitation, and visualization of secreted proteins

Proteins in the cell-free culture supernatant were precipitated by mixing supernatant with an equal volume of ice-cold 20% trichloroacetic acid, washed in ice cold 70% ethanol, then air dried and dissolved in SDS-PAGE reducing buffer (70). Density of the culture (OD600) was determined before preparation of cell-free culture supernatant, and for the

analysis of secreted protein profiles, TCA precipitated protein derived from 5.0 OD600 units

of culture was loaded into the lanes of an 12% acrylamide gel. The gels were stained with Coomassie Brilliant Blue R-250 to visualize the protein bands.

Protein bands were excised using an Ettan™ Spot Picker, and processed for mass spectrometry using a Waters MASSPrep Automated Digestor. Processed protein samples were spotted on MALDI plates and analysis was done using an Applied Biosystems 4700 Proteomics Analyzer. Data were acquired and processed using the 4000 Series Explorer and Data Explorer (Applied Biosystems), and using the MASCOT search engine, the peptide fingerprints were compared to the NCBInr database for Gram-positive bacteria.

2.5.2 Purification of Aur and Geh

For purification of aureolysin, 1.5 liters of RN6390ΔsarA was grown overnight in TSB at 37°C in an orbital shaker, set at 200 rpm and the cell-free supernatant was collected. The supernatant was precipitated by adding ammonium sulphate up to 85% saturation. The precipitate was then resuspended in binding buffer (20 mM Tris-HCl pH 7.4, 5 mM CaCl2)

and loaded on a sepharose packed 5 mL HiTrap column equilibrated with binding buffer. The protein was eluted over a gradient of 0%-80% elution buffer (20 mM Tris pH 7.4, 500 mM NaCl, 5 mM CaCl2). Protein fractions were collected and dialysed against 20 mM Tris pH

7.4 with 5 mM CaCl2. Protein purity was confirmed using SDS-PAGE, and protease activity

was confirmed with a protease assay (described above).

The full length of the gene encoding Geh was PCR-amplified from S. aureus strain USA300 using forward and reverse primers containing NdeI and EcoRI restriction sites, respectively. The amplicon was digested with NdeI and EcoRI and cloned into NdeI-EcoRI- digested pET28a(+) (Novagen), which incorporates a thrombin-cleavable His6 tag at the N-

terminus of the encoded recombinant protein. The pVVgeh (pET28 with Geh) construct was

introduced into E. coli BL21 (DE3). Cells were grown in LB media containing 50 μg/mL kanamycin at 37°C in an orbital shaker, set at 200 rpm. When the culture reached an OD600 of

0.6, isopropyl β-D-1-thiogalactopyranoside was added to a concentration of 0.5 mM. The culture was further incubated for another 16 hours at 25°C, after which cells were collected via centrifugation. The cell pellet was re-suspended in 30 mL Buffer A (10 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole). The cells were lysed using a cell disrupter (Constant Systems Inc.) at 30 psi after which 10 μM E64, a cysteine protease inhibitor, was added to the lysate, and further cell debris was removed by centrifugation (3000 rpm for 15 minutes).

Any remaining cellular debris was removed by ultracentrifugation (50000 rpm for 60 minutes) and the soluble lysate was applied to a nickel-loaded 1-ml HisTrap column (GE Healthcare) equilibrated with buffer A. The His6-tagged protein was eluted from the column

with a gradient of 0%–80% buffer B (10 mM Tris-HCl pH 8.0, 300 mM NaCl, 500 mM imidazole) using an ÄKTA FPLC (GE Healthcare). Protein fractions were collected and dialysed against 10 mM Tris pH 8.0. Protein purity was confirmed using SDS-PAGE. Lipase activity was confirmed using the pNPP based assay as described previously (34) and the protein was quantified using a Bradford Assay.

2.6 Assays

2.6.1 Lipase Assay

Lipase activity was assayed with para-nitrophenyl palmitate (pNPP) substrate (Sigma). For assessment of lipase activity in cell-free supernatants, cultures were grown for 18 hours and filtered across a 0.22 micron filter. Supernatant was concentrated using Amicon 10 K centrifugal filters by centrifugation for 30 minutes 3000 rpm. Prior to assay, the supernatant samples were normalized by dilution with sterile water as needed, to adjust for minor differences in cell density of the stationary phase cultures at time of harvest. Solution A (795 μM pNPP substrate in isopropanol) was added to solution B (0.005 % Triton X-100, 50 mM Tris-HCl pH 8.0, 1 mg/mL gum arabic) at a 1:9 ratio. Aliquots of the normalized supernatant were added to the assay buffer (Solution A and B). For measurement of activity of purified proGeh or mature Geh (as described previously), aliquots of enzyme were added to the assay buffer. Samples were incubated for 30 minutes at 37°C in the dark and absorbance was quantified at 410 nm using a Varian Cary 50 spectrophotometer.

2.6.2 Protease assay

Total protease activity of culture supernatants, as well as purified aureolysin, was assayed with FITC-casein substrate (Sigma). Before the assay, the supernatant samples were normalized by dilution with sterile water to adjust for minor differences in cell density of the stationary phase cultures at time of harvest. For purified aureolysin, aliquots of the protease were mixed in sterile water to a volume of 490 μL. These samples were mixed with 460 μL of incubation buffer (40 mM Tris-HCl pH 7.4, 300 mM NaCl, 20 mM CaCl2, and 2 mM L- cysteine) and 50 μL of 0.2% w/v FITC-casein. EDTA was used as an aureolysin inhibitor. The samples were incubated at 37°C for 2 h in the dark. Trichloroacetic acid was then added to 4% w/v to stop the reaction, and the samples were centrifuged at 15000 rpm for 15 minutes to pellet undigested casein. The supernatant was then mixed with an equal volume of 0.5 M Tris-HCl, pH 8.5, and after transfer to Optilux black clear bottom microtitre plates (BD Falcon), fluorescence was quantified on a Biotech plate reader using excitation at 485 nm and emission at 535 nm.

2.6.3 Aureolysin-Geh processing

The rate of Geh processing by aureolysin was determined to optimize the minimum quantity of aureolysin required to produce mature Geh. Briefly, 3 μg of Geh was first incubated at 37°C for 3 hours in lipase buffer (50 mM Tris HCl pH 8.0) containing varying amounts (1 - 0.001 μg) of aureolysin. Additionally, a suitable time frame for the reaction was determined by incubation of 3 μg of Geh at 37°C in lipase buffer (50 mM Tris HCl pH 8.0) with 0.01 μg of aureolysin. Samples were removed from the incubator at varying time points (5-90 minutes). Reactions were stopped with the addition of SDS-PAGE reducing buffer,

followed by heating for 5 minutes at 95°C. Assessments of the levels of proGeh and mature Geh were made visually following SDS-PAGE.