Heterologous expression of transmembrane protein

Shinhan Shiu sshiu at students.wisc.edu
Fri Mar 7 15:57:29 EST 1997


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
*********************************************



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