Extraction of protein from fungal mycelium: water soluble fraction

Introduction

The following is a method to extract mycelium-associated or intracellular protein from fungal mycelium

Materials

• Fungal conidia stock solutions
• modified Vogel's liquid medium: Vogel's salts in 1% glucose
• Lysing buffer: 100 mM Tris-Cl (pH 7.5), 1 mM EDTA, 5 mM dithiothreitol, 5 mg/ml Pepstatin A in DMSO,1 mM Pefabloc SC
• 0.6 M MgSO₄
• 70 % (v/v) alcohol

Equipment

• Shaking incubator set at 37°C
• 250 ml conical flasks
• Vacuum pump
• Side-arm 1000 ml conical flask
• Buchner funnel
• Whatman No.54 filter paper
• Universal tubes
• Freeze-drier
• 1.5 ml microcentrifuge tubes
• Digital pipette 200 - 1000 µl
• 50 ml polyallomer centrifuge tubes
• MSE High Speed-18 Centrifuge (or equivalent)

Preparation

Sterilize the following materials and equipment: pestle and mortar, 1.5 ml microcentrifuge tubes, polyallomer centrifuge tubes (can be rinsed with 70% alcohol if preferred).
Prepare 50 ml 1% (w/v) glucose solutions in conical flasks, sterilize and add Vogel's salts
Lysing buffer can be prepared in advance, filter sterilized, aliquoted and stored at -20°C

Procedure

1. Inoculate 50 ml of modified Vogel's medium with 10⁵ conidia per ml and incubate cultures using a shaking incubator set at 37°C and 200-250 rpm
2. Collect mycelium onto Whatman No.54 filter paper using a vacuum, side-arm conical flask and Buchner funnel, rinse with 0.6 M MgSO₄ or 100mm Tris-HCl, 1mm EDTA pH 7.5 and remove excess liquid by sandwiching between layers of dry filter paper
3. Freeze-dry mycelium for a minimum of 12 h)
4. Crush freeze-dried mycelium using pestle and mortar and add lysing buffer
5. Centrifuge at 16-18000 x g, 4°C, 15 min
6. Collect supernatant and filter sterilize

Timetable

Tips and general comments

1. The age of the culture used to extract protein depends entirely upon the experimental aims.
2. Pelleting of the fungal culture during growth may pose a problem depending upon the proteins being studied. Pelleting results in a dual-phase culture (actively growing at the outer surface of the pellet, but dying within). If necessary use either an anti-pelleting agent such as Junlon or thin layer liquid culture ( see laboratory protocols 1).
3. Crushing the mycelium is considerably easier if it has been freeze-dried. However, if freeze-drying is not an option, the mycelium may be crushed in a sterile pestle and motar using liquid nitrogen. Ensure that the mycelium is as dry as possible, as too much water makes the mycelium very difficult to crush.
4. The volume of lysing buffer added to the crushed mycelium depends upon the amount of fungal material: 1-5 ml is generally used in this laboratory
5. Filter sterilised protein may be aliquoted and stored at -20°C

References

Hearn VM, Wilson EV, Mackenzie DWR. Analysis of Aspergillus fumigatus catalases possessing antigenic activity. Journal of Medical Microbiology, 1992; 36:61-67.

López-Medrano R, Ovejero MC, Calera JA, Puente P, Leal F. An immunodominant 90-kilodalton Aspergillus fumigatus antigen is the subunit of a catalase. Infection and Immunity, 1995; 63:4774-4780.