I am working with a protein which is told to be secretory and has been purified under native conditions. But When I expressed and induced, it formed inclusion bodies( white preicipitate after sonication). However, initially I ahve been managed to express and purify(Ni-NTA chromatography) it in native conditions, but now I am not able to reproduce the results. What may be the reason.
This protein are expressed in E coli??? If yes the probability of secretion is lower. In this case IB they can be solubilizates for NTA-Ni purification (the column should equilibrate with a same solubilization buffer, in elution remove the solubilization agent). regards
Whether or not the protein forms inclusion bodies may depend on the rate of expression. It sometimes helps to lower the temperature of the culture upon induction, anywhere down to 16oC.
If something is different, previously soluble now precipitating, you MUST have changed and experimental condition. Carefully look at temperature, salts and buffers present, reducing agents and the growth conditions of the cells. Shaking faster, less culture in the flask, slightly higher summer time room temperature, richer media can all cause cells to slip into inclusion body formation. Carefully reproduce your previous conditions, its an essential skill in bench science! The fact that it worked before makes it all worthwhile, good luck!
Hi Kanti
Is it secretary or intra cellular?. Secreatory proteins normally will come in soluble form and it is not need for sonication.
If it is intracellular, expressing eukaryotic protein in E.Coli there may be a chance of forming inclusion bodies if it is overexpressed.
May be you can try at slow rate of induction to eliminate the inclusion bodies but the expression will be less, or you may choose to solubilize the large amount of inclusion bodies in Urea to optimize a new process.
May be this information useful for you. GOOD luck.
Use lower concentration of IPTG during induction and incubate IPTG-induced cells at 15-18C.
Hi everyone
I have a question >> my protein fuse with signal peptide(phoA), when I'm express at 37'C for 3hr after added 1mM IPTG. it can not export to periplasm of E. coli.
Can anyone suggest, What can be the problem? My protein too big?
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