Pichia pastoris Cell Lysis

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sciencechick
sciencechick's picture
Pichia pastoris Cell Lysis

Hi Everyone,
I am trying to lyse my yeast cells (pichia pastoris).  I have tried with a homogenizer and had some luck, I just don't think that I am getting complete lysis and I've done multiple passes.  Does anyone have any experience with Pichia cells in particular and the best way to lyse them?  My end goal is to purify a membrane protein from the prep.  I've heard that Zymolase is good but that it cuts the protein, so I'd rather not use that.   Any information would be appreciated.
Thanks!

Chin Fen Teo
Chin Fen Teo's picture
 Hi sciencechick,

 Hi sciencechick,

Below is the method section adapted from: Yang G et al., Biotechnol Lett (2006) 28:1581–1586.

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mso-bidi-font-family:AdvPSTIM10-R">Cell suspensions were lysed in lysis buffer (50 mM sodium phosphate, pH 7.4, 1 mM 
mso-bidi-font-family:AdvPSTIM10-R">EDTA, 5% (v/v) glycerol, and freshly made protease inhibitors, 1 mM PMSF, 10 mM benzamidine, 
mso-bidi-font-family:AdvPSTIM10-R">20 ug aprotinin/ml, 20 ug leupeptin/ml, and 10 ug pepstatin/ml) containing an equal volume of ice-cold, acid-washed glass beads (425–600 um, Sigma). The mixture was vortexed eight times for 30 s each time with an interval of 30 s on ice and centrifuged at 1500 g for 5 min, then the supernatant was collected and ultracentrifuged at 100,000 g for 1 h (4 deg). The crude membrane pellet was resuspended in solubilization buffer (50 mM Tris/HCl, pH 7.4, 500 m
7.0pt;font-family:Arial;mso-bidi-font-family:AdvPSTIM10-R">M NaCl, 1.0% octyl-b-
font-family:Arial;mso-bidi-font-family:AdvPSTIM10-R">D-glucopyranoside, with freshly made protease inhibitors and incubated at 4 deg for 2 h with stirring. The membrane suspension was ultracentrifuged at 100,000 g for 1 h.

Hope this helps. Good Luck!