Membrane protein - (May/28/2008 )

I’d like to know suitable vectors and E.coli strains to express & purify of hydrophobic proteins (Plasma membrane binding proteins; hydrophobic 50%, hydrophilic 30%, MW: 20kDa, 45kDa).
If it would be impossible, please tell me other suitable system. (But I can not use so expensive systems…) It’s first time for me to purify membrane protein.
Thank you so much for all your help!

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See if you find something here:
Protocols: http://search.vadlo.com/b/q?sn=158621799&a...+coli&rel=0
seminars: http://search.vadlo.com/b/q?sn=158621799&a...sion+&rel=2

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Dear Cellcounter,

Thank you so much for your grate help. I can learn a lot in these sites!

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Our Lab uses the pBAD system from invitrogen...it has much tighter control.
We use BL21 BL21AI C41 C43 and LMG194 Cells as needed.
Good luck.

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We do membrane proteins in BL21 (DE3) strains C41 or C43, which appears to have a more-complex membrane system than regular BL21. We have tested autoinduction media to stop unintended induction, and it works better or worse than standard protocols in a protein-dependent manner.

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Thank you so much for your information. I did not know the LMG cell. And also I am surprised that pBAD system is useful for membrane protein. Thanks a lot!!

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Thank you so much for your wonderful advice! I read some papers about C41/C43. In your research, are there any membrane proteins in a includion body fraction? How about? Is it useful to get 'gram weight' protein?
Thank you for your help.

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Hi,
As far as I know, we don't have an issue of inclusion bodies for our proteins, but we try to keep the expression rate fairly low with low-copy number plasmids. The question of 'gram weight' expression is related to a number of factors, including the codon bias, expression temperature etc. You'll just have to try the system with your protein.

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Thank you for your prompt reply! It's amasing that you don't have any issue of inclusion bodies. You gave me the power to try these methods.
Thank you so much for your help.

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Hi

good luck with ur working, membrane protein are just "sheet" (sorry). i hate them.

u can use almost every system that u got. just remember that such proteins tend to form inclusion bodies

so get some protocols of inclusion bodies purification.

P.S: dont use the pBAD system. i am using it, and it worth... nothing. very little concentrations of the

proteins and u also get a signal peptide of the plasmid attach to ur protein as a bonus.

have lots of fun.

bye

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