S.pombe - disruption

2 posts / 0 new
Last post
Andreas Heinemann
Andreas Heinemann's picture
S.pombe - disruption

We have problems with cell disruption of S.pombe using a FrenchPress high pressure homogenizer.
Does anyone have some experiences in this or other yeast cells?

Tony Rook
Tony Rook's picture
Here's an alternative method

Here's an alternative method that might be helpful...

"Exponential phase cultures of yeast cells (1 litre) were collected by centrifugation at 5000 g for 5 min and the pellets were resuspended in 30 ml of 0.25 M-sucrose/20 mM-Hepes, pH 7.4. Cells were broken with 5 x 15 s pulses of a Bead Beater (Biospec Products, Bartlesville, OK, U.S.A.). The resulting homogenate was centrifuged at 10000 g for 10 min at 4 °C, and then the supernatant was decanted and centrifuged at 105 000 g for 1 h at 4 'C. The 105 000 g pellet was resuspended in the above buffer to a concentration of 15-20 mg of protein/ml as determined by the Lowry protein assay [11]. The yeast cell fractions were either assayed immediately for activity towards
styrene oxide [12] or stored at -70 'C."1

References:

1. MR Jackson, B Burchell. Expression of human liver epoxide hydrolase in Saccharomyces pombe . Biochem. J. (1988) 251, 931-933

Abstract:
Human liver microsomal epoxide hydrolase cDNA was inserted into the yeast expression vector pEVPI 1. The resulting recombinant plasmid was introduced into Saccharomyces pombe. The epoxide hydrolase protein and enzymic activity was subsequently expressed and identified in the 105000 g pellet after centrifugal fractionation of homogenized yeast cells. This method will provide a useful source of human liver epoxide hydrolase, avoiding the problems of obtaining human tissue.