Bacterial strains used in protein expression - (Feb/13/2007 )

Dear all,

What bacterial strains you used to expressed a recombinant protein? I used BL21, the expression was fine but the protein lost its bioactivity. Therefore, I am going to try the other competent cells.
Any suggestions?

Thank you so much!!!!!

siuchi

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what do you mean as "lost its bioactivity"? did it ever had activity as a recombinant protein?

my suggestion goes to XL1-Blue. I had good references before i used it. but dont expect the same amount of expression. but maybe its better to have less since it may result in better refolding.

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lost its bioactivity? are you trying to say that once the protein is being expressed, the protein lost its function?

XL1-Blue? Is it the one with blue white selection?

I used BL21, M15 and SG???? (can't remember the numbers). Maybe you can try out the BL21 Plyss DE3. Cheers

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Hi,
I think that your protein is send in inclusion bodies (you can check it by analyzing the unsoluble debrees on SDS PAGE. I obtained very good yield of protein production using the BL21(DE3)pLysS strain in presence of 0,4 % of glucose and induce the bacteria at minimum OD of 1,5 for 2-3 hours.

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Dear All,

Thank you so much for your kind reply.

My recombinant protein is expressed in periplasmic. Do you think the inclusion body can form in perilpasmic and can be extracted by osmotic stock? Actually, the protein(it is a scFv) could be extracted and purified. However, according to my flow cytometry result, it didn't bind the antigen.

Any suggestion???

Thanks so much!!!!

siuchi

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Hi,
In theory you can extract your protein from the periplasmic space by osmotic shock. I suggest to download the "pET manual" from novagen website, this manual is well documented in terms of protocols and bacteria strains (including strains able to generate disulfide bridges).
Good Luck
TF

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