protein expression in E. coli hosts

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pverma
pverma's picture
protein expression in E. coli hosts

i am trying to express a plant protein in E. coli but till date i could not. although i have cross checked the recombinant clones. Can i know whether the protein gets degraded during expression in the bacterial host? if this is the case then how to prevent its degradation ? Suggest me.waiting
 

kjosephs
kjosephs's picture
How are you detecting

How are you detecting expression?  Is it possible the protein is expressed but it is insoluble?  If it turns out that the protein is expressed and is soluble but rapidly degraged by host proteases (this is sometimes observed be western blot with polyclonal antibody if you have one), the only suggestions I have are to make sure that the purification is conducted on ice or at 4C and to use protease inhibitors like benzamidine and PMSF plus EDTA (if you aren't using nickle affinity for purification).  If you want something a little fancier, I like the protease complete tablets from Roche.

asif123
asif123's picture
Dear,

Dear,
Are you sure about transformation tech?, have you checked the cells for successful transformation? Are you allowing the cells to reach sufficient population generally 0.8 OD at 600nm before induction?
If you have answer in yes for all above questions then rupture the cells and look for inclusion body because generally E.Coli produced protein and peptide in aggregates form.
Regards
Asif