His-tag protein purification - (Nov/16/2005 )
I have tried to express a trucated recombinant cytochrome P450 protein in E. coli BL21(DE3) using Prset as Expression vector. And I have tried to purify this trauncated form by denaturing methods for polyclonal antibodies production. However, both 8M urea or guanidine hydrochloride cannot solubilize the recombinant protein. I have either used sodium phosphate or Tris in my lysis buffer (mainly follow the protocol provided by Qiagen). No matter how long was the incubation time of the lysis process. The recombinant protein keep fall in the pellet fraction.
Can anyone give me suggestions on purification of the recombinant protein under denaturing condition? Thank you very much!
I am not familiar with the protocol you mention. However, when I have resolubilized proteins out of inclusion bodies with Tris/Urea/DTT, I either use a tissue homogenizer or a spatula to break up the inclusion bodies. The appearance of the material changes from opaque to more translucent. In some cases, I have also incubed for periods ranging from an hour to overnight.