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membrane protein expression

expression

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#1 ahmadfathi

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Posted 09 February 2014 - 12:10 AM

hello,

 

i'm doing expression and purification of membrane protein of cytochrome p450

the method for expression as followed:

vector: pCwori

host: dh5a

media: terrific broth

temperature: 30 o

incubation speed: 125 rpm

time: overnight

IPTG: 1mM

lysozyme: 300 ul/g

sonication: 10s burst 15s rest for 5min

 

 

with these established condition i just observed poor band of interest of my targeted protein

any suggestion for me to get overexpression possibly? 



#2 neuron

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Posted 12 February 2014 - 02:45 AM

I don't have any experience in purification of membrane proteins, but in general if the protein goes into inclusion bodies, we try to do the expression at low temperature, i.e. 16 or 18 degree. I don't know if this can work in your case also.

 

I found this product for membrane proteins, it may be useful for you-

 

http://www.lifetechn...ssion-kits.html



#3 labtastic

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Posted 17 February 2014 - 10:23 AM

I am not familiar with the pCwori plasmid, but if it's a T7 promoter based expression plasmid then you need to move it into a BL21 strain for expression.

 

Other suggestions:

Shake your flasks at minimum 200rpm. Use baffled flasks to ensure optimal aeration.

Grow at 37C until OD reaches ~0.8, then drop temps to 18C and grow overnight. It is possible, as mentioned above, that 30C is too warm.

Also, you didn't say how you are assessing expression, just how you lysed the cells. Does your protein have an affinity tag? Run a western blot of non-induced vs induced whole cells probing with appropriate primary antibody (e.g. anti-His). 

If you don't want to do a western, do a small scale purification. Lyse cells as you did, then do a soft spin (10min at 5k x g) to remove unlysed cells, then spin that supernatant at 150k x g for 1 hr to pellet membranes. Remove supernatant, resuspend pelleted membranes in a detergent (1-2% triton for initial expression level testing) and spin at 150k x g again for 1 hr to remove detergent-insoluble material. Add NiNTA (or appropriate affinity resin) to supernatant, wash resin, elute protein, run eluted protein on a gel. 

Also, a "poor band" may not be a bad thing. Membrane proteins don't express as well as soluble proteins. 1mg of expressed protein per liter of culture would be considered very good expression for a membrane protein.

Or try a different expression vector. Try expressing with a cleavable (e.g. TEV) c-terminal (or N-terminal depending on your protein and how it is embedded in the membrane) GFP tag. Expression can increase quite a bit doing this, and determining optimal expression conditions only requires putting whole cells in a fluorimeter and measuring fluorescence. 

 

Most importantly, don't get too frustrated when working with membrane proteins. Not sure exactly what you hope to do with it, but I can assure you that if you're the first person working with this protein it'll be a marathon project.







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