HI Sunil,

Unfortunately (or luckily), all protein are different. Have you codon optimized your gene for E. coli expression?

If not, Codon+ cells lines are worth trying

If yes, then you might want to turn to insect cells or mammalian cells!

Good luck,

Dak.

Hi Daouda,

Thank you for your suggestion. Yes, i am using codon optimized gene for protein expression.

Thanks

Sunil

Thanks Dr. Gedi for sharing your experience. I will try your suggestion.

Regards,

Sunil Singh

Dear Dr. Singh

We have experience with G(GDDD)3 (Rathnaya et al BBA 2013) and G(GRRR)3 both are good. codon are optimized for Ecoli. (GDDD)3 reduced expression in some cases but not always. Best regards,  YK.

Hello Sunil Sing

If your protein is in insoluble form such as inclusion bodies. First you need to use a containing urea or guaniduim chloride buffer, of course evaluating the molarity at which your protein is solubilized. Then a refolding step have to be done. It could be by dilution or dialysis  in order to reduce the concentration of the caotropic agent. In the refolding step its very important to study the pH, protein concentration and some times is necessary to use other compounds such as DTT to improve the recovery.

Best regards, JGS

As a minor (unlikely) suggestions (but may help someone):

1) I had a strange case when protein dramatically improved activity if the saturated E. coli culture in shaking flasks was incubated for 1-2 more days after saturation: https://www.ncbi.nlm.nih.gov/pubmed/23791800. Though, it didn't seem to improve the solubility itself. Could be checked just in case.

2) Some proteins need high ionic strength in the lysis buffer to separate from E.coli debris. Could be checked just in case.

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I also wonder why don't we have some efficient archeal hosts to do protein expression like in E. coli. :( Since many of their cellular systems (including chaperones) are closer to eukaryotic, it could have been beneficial for expression of eukaryotic proteins.