I'm trying to use the Pichia pink strains supplied by Invitrogen to express recombinant proteins. So far I've identified a few clones that express my proteins intracellularly, but the protein yeild is quite low, and I'm yet to find any clones that secrete the proteins.

To screen my clones, I've been following the protocol for pilot-scale expression that is given in the Invitrogen manual. In this protocol, cultures are first grown in BMGY to generate biomass, then switched to BMMY containing 0.5% (v/v) methanol to induce expression. The protocol recommends growing for 24 hours in 1 mL of BMMY containing 0.5% methanol, then adding 100 uL of 40% methanol, which would bring the methanol concentration up to about 4%, and growing for a further 24 hours.

This methanol concentration of 4% seems very high. I suspect that there is an error in the protocol, as everywhere else in the manual refers to using 0.5% methanol for induction. I intend to repeat the screening process on my clones with a methanol concentration of 0.5%, but I was curious to find out if anyone else has had success with growth at 4% methanol.