cleaving protein - (Jun/06/2008 )
Hi,
Im working on a plant protein that is predicted to have a transmembrane helix. I would like to try expressing the protein in
soluble fragment and thus, I have to cleave the protein at its stem (after the helix).
Can someone with experience advise or let me know where I can get information (textbook, papers or experience) to help me decide on the cleavage site,
eg the usual length of a protein stem? And what kinda site should I cleave? How far away from the transmembrane region?
Thanks in advance! Any help is really appreciated as this is my first time dealing with protein work.
Im working on a plant protein that is predicted to have a transmembrane helix. I would like to try expressing the protein in
soluble fragment and thus, I have to cleave the protein at its stem (after the helix).
Can someone with experience advise or let me know where I can get information (textbook, papers or experience) to help me decide on the cleavage site,
eg the usual length of a protein stem? And what kinda site should I cleave? How far away from the transmembrane region?
Thanks in advance! Any help is really appreciated as this is my first time dealing with protein work.
another approach would be to use a detergent to dissolve the membrane? There is a multitude of references about this.
What you would try and do I reckon, your way, would be to clone and express only the soluble portion of the protein?
another approach would be to use a detergent to dissolve the membrane? There is a multitude of references about this.
What you would try and do I reckon, your way, would be to clone and express only the soluble portion of the protein?
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Thanks.
Yeah, thats my intention, to only express the soluble portion of the protein.
Dealing with membrane bound protein might take me more time than i could afford ( as I have to finish my project
by oct). At this point, I am just cloning my genes.
You might consider amplifying just the soluble fragment, which is a standard procedure. www.expasy.org has a number of transmembrane helix prediction algorithms to get yo on your way.
Thanks for ur reply swanny.
I have made the transmembrane helix predictions and got a rough idea of where the transmembrane region is.
Before I can amplify the soluble fragment, I have to decide on where to cleave the protein. I have attached a sketch (sorry bit doggy)
to try express what I mean.
Based on the sketch, Is is usual for the stem of a protein to be
included (thus cleaving at the first red arrow after the helix) or having the stem will possibly interfere with the protein folding; thus having to cleave some of the stem off (arrow lower down)? (N: N terminus, M: membrane). Also what is the general length of a protein stem before it reaches its catalytic domain.Thanks![attachment=4810:protein.bmp]
Thanks for ur reply swanny.
I have made the transmembrane helix predictions and got a rough idea of where the transmembrane region is.
Before I can amplify the soluble fragment, I have to decide on where to cleave the protein. I have attached a sketch (sorry bit doggy)
to try express what I mean.
Based on the sketch, Is is usual for the stem of a protein to be
included (thus cleaving at the first red arrow after the helix) or having the stem will possibly interfere with the protein folding; thus having to cleave some of the stem off (arrow lower down)? (N: N terminus, M: membrane). Also what is the general length of a protein stem before it reaches its catalytic domain.Thanks![attachment=4810:protein.bmp]
sorry I posted 2 similar replies. Thought I could get rid of the 2 similar attachments by editing it.
Thanks for ur reply swanny.
I have made the transmembrane helix predictions and got a rough idea of where the transmembrane region is.
Before I can amplify the soluble fragment, I have to decide on where to cleave the protein. I have attached a sketch (sorry bit doggy)
to try express what I mean.
Based on the sketch, Is is usual for the stem of a protein to be
included (thus cleaving at the first red arrow after the helix) or having the stem will possibly interfere with the protein folding; thus having to cleave some of the stem off (arrow lower down)? (N: N terminus, M: membrane). Also what is the general length of a protein stem before it reaches its catalytic domain.Thanks![attachment=4810:protein.bmp]
I don't think there should be a problem leaving the stem on. You could always try making both the short and long versions and see if there's any difference.
I also don't know if there's a generic or indicative length of the stem prior to the catalytic domain. You can test this in expasy under a domain searching algorithm to see where the active domain sits.
Thanks swanny!