Membrane Protein Extraction - (May/09/2013 )

Hi,

I am expressing a membrane protein in E coli and I am having trouble extracting it. I have tried 1% dodecyl maltoside, 1% DDM + 0.5% CHAPS, and 1% DDM and 0.5% sodium cholate. I have worked with membrane proteins from Sf9, and 1% dodecyl maltoside works. Additionally, another membre in my group uses 1% DDM with an E coli protein and it works great.

I first lyse the cells then spin down the inclusion bodies at 10,000xg for 15 min.

Then, I centrifuge the supernatant at 50,000xg for 1 hour to get the membrane fraction.

I homogenize this in the detergent/glycerol buffer, but this is where I get stuck. The protein is expressed well, but I can't get it out of the membrane.


Any suggestions?

are you sure it's not in the inclusion bodies?

if it's foreign to e. coli then it may not have incorporated into its membrane.

The first question is: how do you know your protein is expressed?

I know this may seem like a lot of work, but when working with membrane proteins I recommend expressing with a c-terminal GFP tag (ideally, superfolder GFP). After expression, you can check fluorescence of whole cells (no lysis required) to see if and how much protein is expressed. Expression conditions can easily be optimized this way. Once expression is optized, lyse the cells, centrifuge as necessary and solubilize the membranes with various detergents. Check fluorescence at each stage to track where your protein is going and how efficient the extraction is in the different detergents. You will learn a lot about the personality of your protein this way with minimal work and reagents.

Before setting out to do this, make sure the c-terminal end of your protein is cytoplasmic (as opposed to periplasmic), otherwise the GFP fusion will not work. Most membrane proteins I believe have cyplasmic c-terminal tails

To make your life even easier, engineer in a TEV protease site (or any other protease cleavage sequence) between your membrane protein and the GFP tag. This way once you have a handle on your protein's personality, you can can just cleave the GFP and use the protein as needed for experiments without a potentially interfering tag.