Gel Filtration/Protein Precipitation - (Jan/17/2006 )

Hi all
I am trying to express an inherently soluble protein in E Coli. I obtain inclusion bodies, which I solublize with urea and pass it over a gel filtration column. However the fraction which contains the most protein also contains a couple of other bacterial proteins which cause precipitation. I am using Sephadex G-75. Is there any way around it? I am not able to use the protein because its really turbid.

Thanks

B

hello,

does your expressed protein contain any sort of fusion tag (His tag, GST, etc)? If it has a His tag, you can just purify it w/ NiNTA resin in 4M urea (they have protocols for purifications under denaturing conditions...they work fine). alternatively, you can try and change your induction conditions to avoid the production of inclusion bodies. typically, there are a few tricks that work pretty well. try the following: lower the amount of IPTG to 0.5 or 0.1mM and induce for an hour. also, induce at lower temperatures...30C works well in some cases, and in our lab, we've had to induce for 4hr at 15C to prevent inclusion body formation.

if that still doesn't work, you can "go old-school" to desalt your protein sample, and dialyze it overnight. this is probably the easiest technique ever used in biochemistry. still, be careful not to load too much protein in your dialysis membrane, or it might burst. you'll probably want to check which type of membrane (regenerated cellulose, cellulose ester...etc) is stable in the presence of 4M urea.

one last thing you can do might be to express your protein in a different vector system. sure, it might take you a few days to clone/express the protein in a new vector, but you'll probably spend just as much (or more) time screwing around trying to optimize induction conditions and handling inclusion bodies. pMAL vectors (these give you a maltose binding protein (MBP) fusion tag that allows you to purify your protein over amylose resin) work well to keep proteins soluble. we treat this vector as a "fusion tag of last resort." in our hands, when a protein forms inclusion bodies while expressed as an MBP fusion, it's going to make inclusion bodies w/ every other expression vector as well.

i hope this helps. if you have any more questions, feel free to email me.

-jon

jonmike.reed@gmail.com