SWAT for protein expression - problems with Poly Ala/GC Rich (Feb/25/2009 )
Hi,
i want to express a TF with a relatively long Poly Alanine Stretch in bacteria to do GST/His Pulldown experiments. I cloned and started first expression experiments in BL21 cells. Some of my constructs work fine, but those containing the Poly Alanine Stretch seem not to be expressed. Are there any special bacteria out there to deal with more difficult proteins? Already heard of a "Gold" version of those Bl21 cells, but from the manual i dont really get if they are somehow more "competent" in translating difficult proteins...
Any hints/suggestions?
Greetings Tobi
-Tobimpi-
Tobimpi on Feb 26 2009, 12:16 AM said:
Hi,
i want to express a TF with a relatively long Poly Alanine Stretch in bacteria to do GST/His Pulldown experiments. I cloned and started first expression experiments in BL21 cells. Some of my constructs work fine, but those containing the Poly Alanine Stretch seem not to be expressed. Are there any special bacteria out there to deal with more difficult proteins? Already heard of a "Gold" version of those Bl21 cells, but from the manual i dont really get if they are somehow more "competent" in translating difficult proteins...
Any hints/suggestions?
Greetings Tobi
i want to express a TF with a relatively long Poly Alanine Stretch in bacteria to do GST/His Pulldown experiments. I cloned and started first expression experiments in BL21 cells. Some of my constructs work fine, but those containing the Poly Alanine Stretch seem not to be expressed. Are there any special bacteria out there to deal with more difficult proteins? Already heard of a "Gold" version of those Bl21 cells, but from the manual i dont really get if they are somehow more "competent" in translating difficult proteins...
Any hints/suggestions?
Greetings Tobi
If you have a long stretch of a single amino acid, try to change the codons occasionally, so you don't get a local exhaustion of tRNAs. Check out this figure from the initial human genome paper out of Nature (2001):-
http://www.nature.com/nature/journal/v409/...9860a0_F34.html
As you can see the system is a bit complex (what a surprise...not), but I think the take-home message is to balance out your codons. All the best with it.
-swanny-