I am trying to purify a new recombinant protein from e.coli. After I purify the protein and run on an SDS gel, I get the correct sized bands. When I run the sample on the FPLC, I get a vary small peak where the appropriate protein should be, and I get a very giant peak showing a mass of protein aggregate. It is too big to be a simple dimer or tetramer, this is protein aggregate.
Here is the basic protocol for Ni-NTA column purification:
- Innoculate and grow to OD600 - 0.8
- Overnight 16C induction
- Spin down cells and resuspend in lysis buffer.
- Freeze overnight in -80C
- Thaw and lyse using a french press
- Centrifuge at 14,000 for 45min (after this, the cell pellet has almost dissapeared, there is a tiny brown/grey pellet... I am guessing this means I have no inclusion bodies)
- Bind and elute to Nickle Beads
- Run product on SDS gel and FPLC.
I do not believe that there are inclusion bodies forming because I cannot see them in the cell pellet after centrifuging. I believe this means that the proteins are soluble within the cell.
Can someone please tell me what problem I am having? What direction can I take in trying to troubleshoot this problem?
Hi if you're not planning for a soluble expression of your protein, then you might go for induction at 37°C to boost up the expression of your protein of interest...you mgiht renature it later by dialysis, G25 or gel filtration. It will increase your protein yield.
You may also try a screening test foe protein induction for smaller cultures at different temperatures and then verifying on gel by Blue Coomassie to see in which fraction you find your protein (soluble or inclusion bodies).
Good luck
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