Testexpression in Pichia pastoris

Materials

These reagents are important:

• YPD – medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))
• BMMY medium (yeast extract (10 g/L), peptone (20 g/L), yeast nitrogen base

(13,4 g/L), potassium phosphate pH 6.0 (0,1 M), biotin (0.4 mg/L), methanol (0,5 %))

• MeOH abs.
• Transformed Pichia clones
• 50 mL falcons
• Spectrometer for OD measurments
• Cuvettes
• 1,5 mL Eppis‘
• PBS
• 4x SDS loading buffer

Procedure

• Inoculate 5 mL YPD medium with one big colony
  • in 50 ml falcon tube (secure lid with tape, don’t tighten lid)
• Grow o. n. 30 °C, 200 rpm
• Centrifuge at 2000 g, 5 min
• Resuspend the complete pellet in 5 mL BMMY medium
• Measure the OD
  • Use a 1:100 dilution for measurement
• Start growth
  • For starters you don’t need to dilute your suspension
  • For optimization purposes you may want to adjust your starting OD
    • Recommended is a starting OD between OD 3 and OD 40
• Grow at 30 °C, 200 rpm
• Take samples of 200 µL and add 25 µL MeOH after 24 h, and 48 h
  • Sample handling
    • Measure the OD (1:100 dilution recommended)
    • Centrifuge 5 min, 2000 rpm
    • Split into pellet and supernatant
    • Resuspend the supernatant in 200 µL PBS
    • Mix supernatant with 4x SDS loading buffer
    • Mix equivalent of OD 1 from pellets with 80 µL of 4x SDS loading buffer
• End the testexpression after 72 h, take samples

For optimization, you may want to try different methanol concentrations between 0,5 % and 1.5 % MeOH