overexpression of cytosolic protein on cell surface

Itisam Sarangi via methods%40net.bio.net (by itisam.sarangi from gmail.com)
Tue Dec 20 00:49:45 EST 2011


Hi,
I have one problem. it might be a becoz of some  fundamental problem.
I want to express some cytosolic proteins (1000 amino acids) on cell
surface. so i can use these for my expts.

Construct design :
 N-terminus flag tag, a signal peptide ( from ICAM molecule)  followed by a
cloning site and a trans-membrane domain with a short tail (from ICAM) and
2A- GFP.

there are lot of glycines in between to help for proper folding. i use 10+
amino acids more on either side of trans-membrane domain.


problems:
1.Without the signal peptide but with trans-membrane domain, i see flag tag
on surface of GFP +ve  cells but with signal peptide no flag tag staining.
which is understandable as signal is chopped off along with flag tag. hence
no staining.

2. when expressed the gene of intererst, with out signal but with membrane
i donot see the expression of protein in gfp +ve cells.


The probbable reasons
->  proteins are toxic hence degraded.
-> signal peptide not strong to guide protein to membrane. any other strong
signal peptide or change in position of signal peptide in construct.

If any one has any experince/ suggestions are welcome


thanks
-- 
With regds

Itisam


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