Insoluble recombinant protein is expressed also with out IPTG - (Apr/02/2012 )
Dear all,
I am trying to express His-tagged 45 kDa protein in E.coli BL-21 cells. My problem is that most of the protein seems to go to the pellet. It seems that the basal level of protein expression is quite high because the 0.5 mM IPTG induction does not seem to increase the protein expresssion so much from the control.
My questions is that do you think that the resason for possible inclusion body formation come from the fact that protein is expressed also with out IPTG induction.
Usually this protein has more strong expression than my other proteins in same vector, which were more soluble.
Cheers!
Hi,
It is possible that your protein is getting expressed without IPTG induction if you see the same 45kda band in your control i.e. in without induction lane. I have experienced the same thing where the size of my protein was around 32kda and it use to express without IPTG induction. We further confirmed the identity of this protein using MALDI.
Neuron
Hola, BL21 is a lacY positive strain and tryptone is a triptic hidrolizate of milk casein, so could contains lactose molecules, natural inducer. Moreover the levels of cAMP are high if you dont have added glucose in the medium. For me this is the explanation of induction from the begining. The fact that this protein is insoluble against others soluble, is the own nature of the protein. Try to add 2mercapto-ethanol 10mM, compatible with the Ni-agarose column, and increase lysis buffer volume in order to try have soluble protein at the lysis moment. If you don´t correct the problem study the isoelectric point of your protein, for changing pH and increase solubility. Buena suerte
Hi
Thank you for the replies. I will try the adding of the 2mercapto-ethanol and increasing the volume of the lysis buffer. I am using phosphate buffer (pH 7.4) and my theoretical pI is 5.86 (according to http://web.expasy.org/protparam/). Do you think it is good?
Hola again, Yes it´s good but don´t worry to get pH8, in this way you have changed reducing agent and pH, both to facilitate the solubility. Buena suerte