3. PURIFICATION AND BIOPHYSICAL CHARACTERIZATION OF SECRETED
3.2 OPTIMIZATION OF PROTEIN EXTRACTION
3.2.2 Materials and Methods
A fast protein liquid chromatography system (FPLC) equipped with OS2 software and chromatography materials including high-substituted ion exchange column with DEAE column or Superdex (10/300 GL gel filtration column) was sourced from GE Healthcare Amersham Biosciences (UK). Dextran blue 2000 was sourced from Amersham Biosciences (UK).
Ampholytes 3-10 and IEF standard markers were obtained from Biorad, Germany. N- Glycosidase F (PNGase F) enzyme was purchased from New England Biolabs (UK). BCA kit was purchased from Novagen (Germany). Pre-stained native, unstained protein standard,
80 already-cast native PAGE and SDS-PAGE polyacrylamide gel were sourced from Invitrogen.
Silver staining (The SilverQuest™) and SimplyBlue™ Safe Stain staining were bought from Invitrogen. CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate), ammonium persulfate (APS), TEMED (N,N,N,N‟N‟-Tetramethylethylenediamine), Fast Garnet GBC Salt, Dithiothreitol (DTT), pyrophosphate, adenosine triphosphate (ATP), adenosine monophosphate (AMP), para-nitrophenyl phosphate (pNNP), phospho-L-serine, phospho-L- threonine, phospho-L-tyrosine, ethylenedinitrilotetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), polyoxyethylenesorbitan monolaurate (Tween 80), acrylamide were all purchased from Sigma (UK). Poly-(vinylidene difluoride) membranes (0.22-0.45 µm pore size) and Viva spin ultracentrifuge were obtained from Millipore (Germany). Protease cocktail inhibitors were bought from Sigma.
3.2.2.2 Lichen collection and preparation
Fresh material of Cladonia portentosa (Dufour) Coem was collected in various sites in the UK:
Peak District National Park, Derbyshire, England and Thurso, Scotland. After collection, the samples were air dried under low temperature (15±2 ºC) on the desk and then stored at -20 °C until used. Prior to experimentation, intact lichen thalli were rehydrated overnight in water- saturated air (over water in a desiccator) at 10 °C, and then saturated by spraying lightly with deionized water. This was to allow the lichen to become hydrated without cell rupture and leakage of solutes which can occur if uptake of water in a desiccated state is too rapid (HOGAN et al., 2010a). Hydrated thalli were then cleaned to remove debris. Glassware used in this study was washed with distilled water using phosphorus-free soap and acid (<10 % HCL), and thoroughly rinsed with distilled water.
3.2.2.3 Acid phosphatase assay/s Acid phosphatase assay A
The routine acid phosphatase activity was determined using p-nitrophenyl phosphate (p-NPP) as substrate, using a method described by TURNER et al., (2001) and optimized by HOGAN et al., (2010a). Briefly, samples of intact C. portentosa were added to 2.9 ml of 0.02 M citric
81 acid-trisodium citrate (CASC) buffered assay medium made up in simulated rainfall containing major ions representative of UK precipitation (20 mM MgSO4.7H2O, 8.0 mM CaCl2.2H2O, 150 mM NaCl, 15 mM NH4NO3, 5.0 mM KNO3) (HAYMAN et al., 2004) and the pH adjusted to 2.5, the optimum value for apase activity in C. portentosa (HOGAN et al., 2010a). Assays were initiated by the addition of 0.1 µl pNPP to yield a final concentration of 3.0 mM, a concentration found previously to saturate apase activity in C. portentosa (HOGAN et al., 2010a). Samples were then placed in a shaking water bath at 37 ºC for 20 min in the dark after which the reaction was terminated by transferring 2.5 ml assay medium into 0.25 ml termination solution (1.1 M NaOH, 27.5 mM EDTA, 0.55M K2HPO4) and the absorbance measured at 405 nm using a NanoDrop ND-1000 spectrophotometer. Thalli were then blotted dry, oven dried for 24 h at 80 ºC and weighed. Enzyme activity was expressed as mmol substrate hydrolyzed g-1 dry mass h-1 using p-nitrophenol (pNP) to calibrate the assay.
In the case of ground samples, the routine assay was modified slightly. The reaction was performed at 37 °C by adding 100 µl of enzyme solution to 2.8 ml of 100 mM CASC buffer and 100 µl 500 µM p-NPP. The reaction was stopped by the addition of 0.25 ml 1M NaOH to the 2.5 ml solution as described above. One enzyme unit was defined as the amount of enzyme which releases 1 µmol of p-NP per min. The amount of inorganic phosphate released was calculated using an acid phosphatase from wheat (Sigma) as a standard.
Acid phosphatase assay B
For substrates other than pNPP or 4NPP, the method of AMES (1966) was used to measure the amount of Pi released by other phosphorylated substrates such as phytic acid, ß-naphthyl phosphate, D-glucose-6-phosphate and ß-glycerophosphate (ß-GLOP). Ascorbic–molybdate solution [2% (m/v) C8H8O8 0.42% (m/v), (NH4)6Mo7O24.4H2O), 1N H2SO4] was prepared as follows: ammonium molybdate (0.42 g) and conc H2SO4 (2.86 ml) were dissolved in 80 ml of dH2O, transferred in a 100 ml volumetric flask and the volume adjusted to 100 ml with distilled water. This solution is stable at room temperature. The needed volume of ascorbic molybdate solution was made fresh in 2% ascorbic acid (the reducing agent) just before use (AMES, 1966). The reaction was measured in 1.0 ml reaction mixtures containing 800 µl of assay buffer
82 (100 mM CSTC, pH 2.5), 100 µl of substrate and 100 µl enzyme. Incubation was carried out at 37 °C for 60 min. The reactions were stopped by the addition of 20 µl 50% (w/v) CASC.
Blanks were prepared in separate tubes by either replacing the enzyme solution with aliquots of buffer and assaying as normal or stopping the reaction at time zero with 20 µl CASC (50%).
After termination of the enzyme activity, 200 µl of the reaction was mixed with 800 µl of ascorbic–molybdate solution (2% ascorbic acid, added just before use) (AMES, 1966). The tubes were incubated for 20 min at 45 °C (AMES, 1966). The inorganic phosphate was measured by monitoring absorbance at 820 nm. The assays were performed in triplicate and controls were run for background amounts of Pi. One unit of acid phosphatase activity catalyzes the liberation of 1 µmol of inorganic phosphate per min. Generation of an inorganic phosphate calibration curve was obtained using a phosphate standard solution which was prepared by making a 1.0 mM stock solution of KH2PO4 in distilled water. This was diluted to prepare a range of phosphate standard concentrations ranging from 200 to 1000 nmol/ml phosphate. The phosphorus calibration curve was performed concurrently with assay B.
3.2.2.4 Determination of protein concentration
Protein concentration was determined by a BCA kit. The test was conducted as per the manufacture‟s instructions. A standard protein solution consisted of Bovine Serum Albumin (BSA) stock solution (1.0 mg/ml) in distilled water. Samples were transferred to clean cuvettes and measured within 10 min at 562 nm.
3.2.2.5 Solubilization of proteins by biological detergents and cell wall hydrolases
The experiment was designed as shown in Table 3.3. The starting material was similar in all treatments, 3.0 g for ground samples and 1.0 g for intact lichens.
83 Table 3.3: Eluting phosphatase with detergents and hydrolase enzymes. Intact vs. mechanical extraction.
N=3.
TREATMENTS CONCENTRATION SAMPLES
Intact thallus Ground
Buffer 20 mM 3 reps 3 reps
Digitonin (w/v) 0.1% 3 reps 3 reps
NDSB 201 1M 3 reps 3 reps
CHAPS 1M 3 reps 3 reps
Glucanex 20 mg/g weight 3 reps 3 reps
Caylase 20 mg/g weight 3 reps 3 reps
Novozyme 20 mg/g weight 3 reps 3 reps
To measure the release of apase in the intact lichen samples, 1.0 g of lichen material of Cladonia portentosa was added to 15 ml detergents (Table 3.3). Materials were shaken gently for 1 h at 50 rpm. A protoplast isolation protocol described by DEBETS and BOS (1986) was adapted for isolation of apase using a hydrolase enzyme. To get whole homogenate, the intact lichen was ground in liquid nitrogen into a fine powder using a mortar and pestle. The ground samples were weighed and 3.0 g of fresh weight was put into a 50 ml flask. Cold MME buffer (1.2 M MgSO4.7H2O, 20 mM MES in 200 ml), 0.26 g (of either enzymes) and 0.08 g BSA were added to each flask. The BSA was added in order to protect the protoplast from fungal /Novozyme protease activity. The treatments were all conducted in three replicates. Reactions were incubated for 1.5-2 h while gently shaken at 30 rpm. To aid visual examination, a small sample (10 µl) was diluted with an equal volume of water (10 µl) and observed under microscopy.
To remove all the cell debris, a purification column made of a 10 ml syringe barrel with pre- autoclaved polyallomer wool was used. After the mixture had eluted, it was rinsed with NM (1.0 M NaCl, 20 mM MES buffer). The mixture was mixed gently and spun at 2500 rpm, 4 ºC for 10 min. The supernatant was removed and stored at -70 ºC until use.
84 3.2.2.6 Optimizing protein concentration
Protein samples obtained in this study required to be concentrated before subjecting them to further purification or SDS-PAGE analysis. Several methods were used to concentrate the proteins: sucrose, PEG, ammonium sulphate precipitation and Viva tube concentrator (Millipore, Germany).
After protein extraction, the clear fractions were divided and 100 ml of crude extract was used to determine an efficient protocol to concentrate the proteins before purification. In the case of freeze-drying treatment, 500 ml bottomless flasks were used, where the liquid was gradually frozen with liquid nitrogen by tilting the flask of liquid nitrogen, until all the liquid had turned to ice. Samples were freeze-dried overnight if the volume of 100 ml was used.
The dialysis tube was used to concentrate proteins by either PEG or glucose. The dialysis tube was initially boiled for 10 min and fractions were added to the dialysis bags. After sealing the samples, the bags were placed in a container consisting of dry glucose granules. For PEG treatment, dialysis bags were place in the liquid that consisted of 100 % (w/v) PEG solution.
Solid ammonium sulphate (NH4)2SO4 was added to the enzyme preparation where 40% (w/v) (saturated) was added to the solution and gently stirred for 10 min and centrifuged (12 000x g, 4 ºC, 30 min).
For Viva spin columns, tubes came in 10 ml and 5.0 ml sizes. The protein samples were placed in the sample container and the Viva tubes were centrifuged at 1000x g at 4 ºC. Once the sample had been sufficiently concentrated (5-10 fold, 2-6 h), both flow through and retentate were assayed for apase activity and protein concentration as described in Section 3.2.2.4 to assess the solute recovery.