Materials and methods

Fungal cultures, induction of enzyme activities and harvesting of supernatants Trametes versicolor strain number 6 from the collection of the Institute of Forest Botany, Georg August Universität, Göttingen were cultivated on basidiomycetes-medium (BSM, Hüttermann and Volger, 1973) with 1.5% agar. Starter liquid cultures were prepared by inoculation of 100 ml BSM liquid medium in 500 ml conical flasks with three agar pieces (1 cm in diameter) of the stock culture and cultivated stationary at 25°C in the

Chapter 3. Native staining

dark. Mycelium of 14 days-old liquid cultures were gently disrupted using an Ultra-Turrax T25 (rod diameter 10 mm; Janke & Kunkel, IKA-Labortechnik, Germany) at 9,000 rpm for 1 minute under sterile conditions. Each 4 ml of mycelium suspension were used to inoculate new 100 ml BSM liquid cultures. Standing cultures of T. versicolor were induced after seven days with 100 µl 0.5 M 2,5-xylidine and 100 µl 150 mM CuSO4 respectively of incubation and incubated further for seven more days stationary at 25°C in the dark. The culture supernatants were harvested by vacuum filtration using filter paper (595 Round filter, Ø 90 mm, Schleicher & Micro Science GmbH, Germany).

Coprinopsis cinerea AmutBmut was grown in YMG/T media (Granado et al. 1997) supplemented with 0.2 mM copper and cultures kindly provided by M. Navarro-González. Starter liquid cultures were prepared by inoculation of 100 ml YMG/T liquid medium in 500 ml conical flasks with three agar pieces (1 cm in diameter) of a stock culture and cultivated stationary at 37°C in the dark. Mycelium of five days-old liquid cultures were gently disrupted using an Ultra-Turrax T25 at 9,000 rpm for 1 minute under sterile conditions. Each 1 ml of mycelium suspension were used to inoculate new 100 ml YMG/T liquid cultures. Standing cultures were grown at 37°C for 3 days and supernatants were harvested as above.

Native protein sample preparation

The supernatant obtained from 14 days old cultures was centrifuged for 20 min at 3,000 g and was concentrated by ultra-filtration with 3,000 MWCO membrane (Vivaspin, Vivascience AG, Hannover Germany) at 3,000 g to obtain at least 20 U/ml concentration of laccase activity. Laccase activity was measured by oxidation of ABTS in spectrophotometeric test at 420 nm for 5 minutes as described by Matsumura et al.

(1986).

Native 1D/2D-PAGE and in-gel staining of native phenoloxidases

1D-PAGE electrophoresis was performed using 12x12 cm discontinuous 12%

acrylamide gels with 0.025 M Tris base, pH 8.3 as electrode buffer (Laemmli 1970).

Protein samples were mixed in sample buffer (0.06 M Tris-Cl, pH 6.8, 10% glycerol,

Chapter 3. Native staining

0.025% bromphenol blue). 20 µl samples corresponding to 6-10 mU laccase activity were loaded and run at 10-15°C at a constant current of 30 mA.

For 2D-electrophoresis, protein samples were dissolved in loading buffer [8 M urea, 4%

(w/v) CHAPS, 50 mM DTT, 5% (v/v) Triton X100, 0.67% (v/v), ampholyte buffer (Amersham Biosciences)]. 350 µl of sample solution containing approximately 80-100 µg protein was loaded on 18 cm IPG-strips, pH range 3-10 (Amersham Biosciences) in ceramic holders, rehydrated and focused using an Ettan IPGphor isoelectric focusing machine (Amersham) for a total of 46,000 Vhr at 15°C with the following settings: 100 µA per strip, (12 hr, 20 V, step and hold), 200 V (1hr), 500 V (1hr), 1,000 Vhr, 8,000 Vhr (8,000 V), 40000 Vhr (8,000 V), all according to the protocol of the manufacture. In the 2^nd dimension, proteins were separated on 12% PAGE using Ettan DALTsix electrophoresis chamber (Amersham) together with a protein marker (Precision Protein Marker, Biorad, 10–250 kDa).

Proteins were separated at 10°C for 5.5 hrs in two steps: Step-1: 600 V, 400 mA for 30 min (low voltage is applied for short time to allow the migration of proteins from the IPG strip on to the gel), step-2: 3000 V, 400 mA, 100 W for 5 hrs (high voltage is applied for longer time to resolve the protein spots). The gels were fixed in 30% methanol with 12%

acetic acid and 0.05% formaldehyde overnight and subsequently stained with silver (Blum et al. 1987). For colloidal Coomassie blue staining, gels were fixed in 12% (w/v) trichloroacetic acid (TCA) solution overnight (Candiano et al. 2004). For native staining, gels were transferred to 100 mM sodium acetate buffer pH 5.0 for 30 min. The buffer was changed by 100 ml of fresh buffer and substrates of interest and combinations of substrate were added and incubated with the gels at room temperature according to need (Table 1). Stained gels were scanned at 300 dpi resolution (Microtek, TMA 1600, Hannover, Germany).

Protein identification by ESI-LC-MS analysis

Stained bands were excised from 1D gels, cut into small pieces, washed once with 200 µl of distilled water for 15 minutes and 200 µl of 50% methanol, and dehydrated in 200 µl of pure acetonitrile for 20 min. In-gel digestion of proteins with trypsin was performed at

Chapter 3. Native staining

58°C as described by Havlis et al. (2003). The resulting peptides were extracted in two steps with 200 µl of 5% formic acid and 200 µl of 50% acetonitrile with 5% formic acid (30 min each step). The pooled extracts were concentrated using vacuum concentrator and peptides were dissolved in 5 µl of 5% formic acid for ESI-LC-MS. 4 µl of sample was analyzed by ESI-LC-MS (Esquire-3000, Bruker Daltonics, Bremen, Germany). Gel eluted peptides were injected into the liquid chromatography columns and the corresponding peptides were passed through a high-voltage electric path for ionization in the mass spectrometer, ESI-LC-MS (Electro Spray Ionization-Liquid Chromatograph-Mass Spectrometer; Esquire-3000, Bruker Daltonics, Bremen, Germany) for mass spectrometric analysis. MS/MS spectra obtained from the ESI-MS detector were further processed by the Bruker Daltonics Data Analysis software (Interactive data processing, esquire series) and exported to the protein database and search engine Mascot (version 2.0, Matrix Science, http://www.matrixscience.com/ search _form_select.html) in Mascot Generic Format (MGF). Searches were performed first against a commercial mass spectrometry data base (MSDB) (ftp://ftp.ncbi. nih.gov/repository/MSDB/msdb.nam). In addition, an own database for Mascot analysis was created (19^th May 2005) with sequences from SNAP predictions and annotated protein sequences from the C. cinerea genome (Jason Stajich, Duke University, unpublished, http://genome.semo.edu/cgi-bin/gbrowse/coprinus) combined with the SwissProt database, the NCBI database, the annotated Phanerochaete chrysosporium proteins, and 17 laccase sequences from C.

cinerea annotated by S. Kilaru (unpublished). All cysteine residues were searched as reduced and methionine residues in the oxidized form. Mascot parameters were set to use monoisotopic masses with tolerance of 1.5 for precursor ions and 0.5 for fragment ions.