Heterologous protein expression in the yeast Pichia

Antony Cooper cooper at molbio.uoregon.edu
Mon Jul 10 11:18:49 EST 1995


I've expressed several secreted proteins in Pichia pastoris through 
the in vitrogen system and the proteins are secreted at acceptable
levels.  The problem is that the proteins when purified appear greeny-
brown in colour when they should not be.  When the culture supernatant
was run over an ion-exchange column, the column turns this colour and it 
elutes throughout the gradient.  When the fractions containing my protein
are dialysed and re-bound to the ion-exchange column, elution causes the 
colour to now only elute in the fractions containing my protein.  SDS-gels
do not identify any other protein in my prep and dialysing does not remove
the colour.  I believe that some compound in the culture media has bound
to my protein and since I'm doing binding assays I'd rather not have 
this occur. This coloured situation has occurred when expressing two
very different proteins.  I've grown my cells in Yeast Nitrogen Base
(Difco) plus or minus Yeast Extract and still see the problem.  Invitrogen
has not been very helpful.  If anyone has had a similar problem and have
found a solution I'd love to hear from you. 



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