Hi Mahbobeh,

From my experience, proteins larger than 60 kDa do not express well in E.Coli. I would recommend using insect cells if you aim to produce a 125 kDa recombinant protein.

I have cloned and expressed a functional lanthionine synthetase which is a 119.6 kDa protein in E. coli BL21(DE3). It unpublished but there are many past publications on the same protein that suggest that it's never a problem. Maybe protein specific, you can give it a try.

Thanks Mr Singh for your encouraging answer.

Best,

Thanks Mr.Nicholas. yeah many research suggest that but i am not sure if its always the case.

Best

Dear Birkedal,

Thanks alot. That was really helpful.

Best.

Mah

Hi Mahbobeh,

to reinforce comments from previous posters above, although you may need to trial different media and induction conditions as well as several expression strains, expression of proteins >100kDa in E.coli is undertaken quite regularly in my professional environment, and although less 'easy' than smaller proteins, it is not something to be wary of.  It is only when you start getting into the really huge mammalian proteins that expression in these systems starts to become more trouble than it is worth.

Please feel free to discuss any thoughts or questions that you may have here further - we're all here to help!

best wishes,  Robin

Dear Robin,

Thanks alot for your respose. I may try it using strain BL21first.

Remember that if contains disulfide bridges you should incl. a signal peptide that direct it to the periplasm. I do this regularly with bacterial proteins in E. coli - also with proteins in 100-150kDa range.

As an alternative you could also consider the BL21 derivative Origami DE3 or similar strains from other suppliers where the redox pathway in the cells has been modified to be more permissive to disulphide bond formation.  Remember that if the protein depends on the periplasmic export pathway this can easily be swamped if the protein is expressed at very high levels and if this happens, you can end up with proteins on which the periplasmic leader peptide (what is normally removed as part of the export process) remains - this can in turn mean that you end up with a mixed population of proteins both with and without the leader peptide.

Best of luck!