Hi Kamlesh.

Which time do you use for expression? I have noticed for my transcription factor, that both temperature and expression time are essential for solubility. For me I found that 4 hours at 30°C are fine, but any longer and it starts to become insoluble.

Furthermore what are your conditions for the solubility test? I usually use lysozyme for small scale samples, but sometimes for some reason the lysis does not work at all, shown by all proteins being in the pellet fraction and near to none in the supernatant.

You are expressing this in bacteria. Most recombinant proteins go into inclusion bodies. You might try room temperature for your cultures. Solubility is one big reason people use His-tagged proteins in E. Coli, since they can be purified on Ni+ columns in a denaturing buffer like 6M GHCl. They are then dialyzed stepwise into your buffer of choice to try and refold the protein. Talk to a biochemist or check out the pRSET vectors and their protocols.

Hi there,

IPTG concentration, length of induction, temperature, cell density at induction are the usual parameters to play with in order to improve your protein solubility. Some other aspects can be considered though : co expression of protein chaperones, use of a solubilising tag like MBP or GST or culture the cells in auto inducible media which allows a progressive induction of protein expression...

I hope it will help...

CONDITION  FOR MY PROTEIN IS :

pET28 and pET 32.... 20 degree tem(0.3 to 0.5 mM IPTG) same IPTG at 25 and 30 degree for 15 hour...expression is very high in all codition. my buffer (pH 8)is 100mM tris +150 mM NaCl. 5 ml of culture was pelleted down in 1.5 ml MCT then resuspended into 1 ML of Buffer.. lysed cell by lysozyme and sonicated it. spin at 4 degree for 10 mins at 10000 rpm.. 20 microltr super natant was loaded where all bacterial protein is there in gel except my protein ..It is Transcription Factor of 36 kDa..

pellete was also loaded after resuspending into 100micro ltr of buffr and spin at 12000 for 1 mins..

Now let me know what to do now