[ccp4bb] Adding a transmembrane segment

[aka akaka]

Dear All

I am working with a viral protein that contains an ectodomain, a TM segment and 
a cytoplasmic region. I was wondering if there is a general strategy that 
allows the addition of the transmembrane segment to the ectodomain without 
compromising solubility and secretion.

Thanks

Dr. R. Depetris
Weill Medical College of Cornell University

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Re: [ccp4bb] Adding a transmembrane segment

[Pascal Egea]

Hi,if you add the TM segment, I would expect that the protein will then get
targeted to the membrane and hopefully inserted correctly. My guess is that
you will have to treat your protein as a membrane protein or a
membrane-anchored protein...which means prepare a membrane fraction, use
detergent and look for your protein in there.
Is your ectodomain N or C-terminal (I assume it is N from your message)?
Have you looked at the targeting sequences present in the protein (signal
sequence, reverse anchoring motifs)?

Hope this helps

Pascal F. Egea, PhD

Re: [ccp4bb] Adding a transmembrane segment

[Artem Evdokimov]

Hi,

 

Could you clarify your request a bit? Generally speaking if you allow the
ectodomain to have a membrane anchoring segment it will 'compromise'
solubility since the resulting protein most likely requires special
treatment (detergents) in order to be soluble. There's also the matter of
the secretion signal - depending on your expression system you may succeed
using the original secretion signal, or you may have to engineer an entirely
new one.

 

Artem

 

Nothing is built on stone; all is built on sand, but we must build as if
the sand were stone 

 Jorge Luis Borges

 

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of aka
akaka
Sent: Sunday, September 06, 2009 8:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Adding a transmembrane segment

 

Dear All

I am working with a viral protein that contains an ectodomain, a TM segment
and a cytoplasmic region. I was wondering if there is a general strategy
that allows the addition of the transmembrane segment to the ectodomain
without compromising solubility and secretion.

Thanks

Dr. R. Depetris
Weill Medical College of Cornell University

  _