induction at 30 degree?? ..seems very high for insoluble proteins. I recommend to start with lower temperature ( may be 16 degree) and lower IPTG concentration too. If these simple trick does not work then you might need to make a fusion tag with maltose binding protein or thioredoxin etc. but there is no gurantee that it will solve your problem ..sorry

Hi Kyle..can you give me some example of vectors which does not allow high expression of proteins? I am also recently working on a protein which is gene optimized and cloned into PkA8H vector. I have a feeling that due to gene optimization, it expresses so much that the cell can not keep up with the soluble protein.

Thanks for the comments.

Since my protein is residing mainly in the cell pellet ( checked via Western blot) and is not present in the supernatant...I shall try the suggested ways..and make more pellets to play around...and if nothing works..will re strt the cloning i guess...rest fingers crossed...

Hello,

I am in the process of optimisation.

I also wanted to ask that how is the TPP ( Three phase partitioning ) method for protein purification from inclusion body. Could anyone recommend or comment about it for the same problem mentioned above.

Thanks,

Kritika:)

Hello all,

Thanks a lot.

The protein was detected in the soluble form in SDS-PAGE gel run by playing around with the induction temperature and concentration.

My protein worked by induction at 20 degrees for 4 hours and 0.5mM-1mM IPTG concentration seems to work for my protein

Kritika:)

Congratulations :)