Dear C. Brovic,
The EGFP sequence has been optimized for human expression by changing the
codon usage to that prefered by mammalian cells. My guess is that your
constructs aren't working because of this. My experience with the wt (A.
victoria) cDNA and the S65T mutant (A. victoria codons) was that even
single copy integrants of an S65T GFP-calmodulin fusion were easily
detectable in both S. cerevisiae and S. pombe by fluorescence microscopy
and by FACS. Cerevisiae cells containing a GAL driven S65T GFP construct
were screaming. Pombe cells containing nmt driven S65T GFP were so bright
they look like little "light bulbs". Brendan Cormack has found that the
A. victoria cDNA is not expressed in C. albicans and he has constructed a
yeast codon usage version of his FACS selected mutants that does express.
Supposedly these versions give a further few-fold increase in budding
yeast, but I haven't tried them myself. So EGFP is probably not thecDNA
of choice for yeast.
Mike Moser Tel: 206-616-7391
UW Department of Pathology FAX: 206-543-3644
Box 357470 moser at u.washington.edu
Seattle, WA 98195 http://weber.u.washington.edu/~moser
On 29 Apr 1997 bozic at britbio.co.uk wrote:
> EGFP in S. cerevisiae
>> We are trying to express and detect EGFP protein in S. cerevisiae.
> Gene sequence has been checked, and Nothern Blot analysis indicates
> that the gene is transcribed, nevertheless, we are not able to
> detect any fluorescence (fluorescence microscope, FACS,
> Fluorometer). We decided to construct a strong positif control (EGFP
> cloned in a multi-copy plasmid, under control of a strong inducible
> promoter, and transformed in a protease deficient yeast strain), but
> as previously, no fluorescence is detected. Cells have been grown on
> liquid and solid medium, at 30C and at room temperature.
>> Has anybody same problems to detect EGFP or has someone some
> indications which could help us ?
>> Thank you for your help.
>> C. BOZIC
>bozic at britbio.co.uk>>