expression in Pichia - (Aug/09/2005 )

Dear all,
I this is a new field for me, and therefore i would like to ask a few questions:
1) I am using Pichia Pastoris with pPic vector. by using 3 and 5 AOX primers, I have checked my transformants. They are Mut+, but I am not getting expression. I have tried from 0.5 to 4 % of alcohol. Sometimes I do get an expression, but contamination with the other proteins is heavy.... Other protein bands are dominant. I tried different conditions, changed media, used media by the other members of the lab, but in vain. Sometimes I don't get even a single band either in preinduction medium or even after 96 hours. I am not able to find out where I am doing wrong.
Condtions for expression:
200 rpm, 29.5 to 30 degree celc. + / without 1% Casamino acids as extracellular protease inhibitor. media: BMGY n BMMY ( 0.5 to 4%).
eagerly waiting for your help

HI


1) I am using Pichia Pastoris with pPic vector. by using 3 and 5 AOX primers
,
which vector u r using pPIC or pPICalpha???

How do u know that the other strong bands are comtaninants (if u r using pPICZalpha vector, this may be ur protein modified by glycosylated)

just check

clone