easy protein extraction from Pichia pastoris - (Jul/11/2006 )
I'm trying to express a 70 kDa protein in Pichia pastoris. I've got the suspect that the protein remains inside the cells (i.e. secretion fails) since I don't see any expression through western blot.
Not even with previous ammonium sulphate precipitation of the super natant.
I'm searching an easy phisycal or chemical protocol to release the total proteinaceous content of cells to test it by western blot.
Any suggestion is more than welcome.
I only have experience with glass-beads. But there are protein kits available with lysis buffer that should work on yeast.
could you please tell me how you proceed with glass beads?
We have those in our lab, but nobody has used them for this purpose.
Protein extraction from P. pastoris
Samples were taken from the expression cultures. These were spun at 4° C at 2500 g for 5 minutes. The supernatant was prepared for SDS-PAGE by adding 1/4th volume of 4x Sample buffer (0.25 Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 20% 2-mercapto-ethanol, 0.04% Bromophenol Blue) and heated for10 minutes at 95°C.
The cell pellets were broken by adding breaking buffer (50mM sodium phosphate, pH 7.4, 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma), 1 mM EDTA and 5% glycerol) and glass beads (0.8-0.11 mm, Roth). This was vortexed for 30 seconds and then put on ice for 30 seconds. This was repeated 8 times. Then this was spun for 10 minutes 4°C 14000 rpm and the sup was prepared for SDS-PAGE by adding 1/4th volume of 4x Sample buffer and heating for 10 minutes at 95°C.