Expression in Neurospora

[biochem at clio.rz.uni-duesseldorf.de]

> : 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.
> 
> and Dan Ebbole replied:
> 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.  

Geoff,
in my opinion this need not to be a promotor related effect.
To constitutively express a protein in N. crassa, I tried both, 
mtr and gpdA promotor using the pBAR expression vectors deposited 
at FGSC. Both didn't work with different Neurospora genes. More 
interestingly, as a control experiment I inserted genomic DNA of 
two genes in pBARKS1 and both genes were expressed under the 
control of their own promotor. Almost all stable heterokaryotic 
transformants showed expression. 
So I can conclude that ectopic integration isn't the problem. 
A promotor related effect seems unlikely too, given the fact that 
none of the three promotors works. I'm currently trying to 
construct some new plasmids with mtr and gpdA promotors and hope
I'll get them to work.
In our lab, we had good experiences with the qa2 promotor (Fecke et al. 
FGNL 40:34, 1993) but induction with quinic acid isn't an ideal system 
for biochemical studies of purified Neurospora proteins.
I would appreciate it very much if we could discuss this problem in
more detail! 

Christoph Kruell
Universitaet Duesseldorf, Germany
biochem at clio.rz.uni-duesseldorf.de