In article <5fpvjq$2pj4 at news.doit.wisc.edu>, sshiu at students.wisc.edu
(Shinhan Shiu) wrote:
> Hi there,
>> I tried to express a Arabidopsis transmembrane receptor in yeast by using a
> CEN plasmid with LEU selection and a GAL1 promoter. Somehow I couldn't
> detect the expression by western.
>> The original vector have a mammalian receptor (FGFR) in it and express fine
> after induction.
>> I checked the sequence of my construct: start codon is there follow by signal
> sequence of the the plant receptor then the rest of it.
>> I also checked the message: no mRNA detectable.
>> My guess is that, maybe the yeast just doesn't like the receptor, i.e. there
> is a toxicity problem. But I got reasonable # of transformants and GAL
> promoter should be pretty tightly controlled (right?)
>> Then, it's possible that the yeast transcriptional machinery can't recognize
> the context surround the start codon of the plant receptor. I am planning on
> making a chimeric protein with the first 20 something aa. of FGFR and
fuse the
> plant receptor together with it. Hopefully this will bypass the problem of
> context.
>> I'd greatly (or GREATLY) appreciate any comment or suggestions.
>> Best Wishes.
>>>>> *********************************************
> Shinhan Shiu
> Department of Botany University of Wisconsin-Madison
> 132 Birge 430 Lincoln Dr, Madison, WI 53706
> tel: 608-262-4008
>sshiu at students.wisc.edu> *********************************************
Dear Shinhan,
What yeast strain did you use ? We experienced similar problems in a w303
strain. Maybe there is a problem with GAL induction in that background ???
Other WT strains seem to work (at least for us).
Best wishes and good luck,
Sepp D. Kohlwein
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Sepp D. Kohlwein, PhD
Genetics and Molecular Biology Group
Department of Biochemistry and Food Chemistry
Technical University Graz phone: ++43 (316) 873-6456
Petersgasse 12 fax: ++43 (316) 873-6952
A 8010 Graz, Austria e-mail: kohlwein at ftugax.tu-graz.ac.at
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