Expression in Neurospora

G Turner mb1gt at sunc.sheffield.ac.uk
Tue May 30 08:06:24 EST 1995


Dan Ebbole (dje0282 at VMS2.TAMU.EDU) wrote:
: Geoff Turner
:  University of Sheffield  found:

: We wish to overexpress N. crassa glutamate dehydrogenase, 
: since we are working on its structure, and E. coli is 
: found to give insoluble, inactive protein.
: We turned to N. crassa, and used the grg-1 expression vector 
: deposited at the FGSC.  Our initial transformants with 
: grg-1 driving the am gene (=gdh) are rather disappointing.

: stable this expression is during subculturing.  With the con-10 gene
: expression is reproducible between independent transformants and over time
: with culturing.  So it looks like there may be some Grg-1 specific problem.
:  I guess these are ectopic transformants and unsure how this might affect
: things.  

Dan,
Thanks for the useful comments.  It might be a promoter related effect, 
but it is also true that our transformants are ectopic.
Our results are also similar to those found by Bull and Wootton,
1984, who also used gdh to transform an am- deletion strain.
However, they used only the unmodified gene fragment of gdh.
They never got up to wild-type gdh activity in their 
transformants.
All the best
Geoff

--
 Geoff Turner
 University of Sheffield



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