Hello,

What do you mean by insufficient expression? The protein which your expressing now is it properly folded?

There are many reasons for low expression.

You can check if your sequence has rare codons, if so you can use Rosetta cells instead of BL21. Or use codon optimized gene in BL21.

Dear Usha Kantiwal

it depends a lot from the properties of your protein. In general several factors may limit the yield of eucariotic proteins in bacterial sistem.

- presence of rare codon in the DNA, that could be solved at lead partially by using Rosetta strains or by using a optimized DNA sequence for cloning in the expression vector

- High MW of the proteins

- Presence of PMT (as e.g disulfide bonds) essential for protein stability that are not formed in the bacterial citoplasm. (in this case expression in Origami strain or addition of signal peptide leader (e.g ompA and or pelB) to localize the expression in the periplasm could be usefull.

Some time also the addition at the N-term of a fusion tag (eg GST, MBP, GB1, TRx) that show hgh expression in E.coli can allow to improve the specific productivity.

there is not a unique universal solution, some can work well for a protein but not for one other.

you can see an overview of all the different tools available for E.coli in the following link

https://www.blogger.com/video.g?token=AD6v5dzPeA9ws3hcPvrO8fBABN8iFQ0dwI7GIHys9p8dwVtov4dGrKXH9PF91d9bVMYcLxTYY3XJesKt62Y-LsZcaHDCJKZbNW-DLUm_4p5-75Dbj1VZlKTlyyl2aQzf-EPwO3Ac6eBG

last but not least, if you have low specific productivity but the protein quality is ok, you can also just to try to improve the volumetric productivity buy using an high cell density medium as Enpresso

https://www.blogger.com/video.g?token=AD6v5dxhZKT6Tvug0rLIZgcg8nyl5CwzQSFiX9pLglN-foSxrhnBjoEr7ReFTWj5bbeiloMCNhNoJn2HgHEgR2eC7SYwl5TPVwlCYNniPhSpeELHsevHk1dREBGIdHqskofd9HgX-2gr

of couse one other possibility is express directly it in mamalian cells, those in the last years show amazing progresseshttps://www.blogger.com/video.g?token=AD6v5dyTuEwDXD2ztCnLIFHkG06cCp1jaRuUHOGFtHdiTFnj-yS_TyCyPqzvi6RogJaSP_sXqbJnMDIYplkXODpfOc21BETPPh74J7FJuwMPxHW4X79mURcAl7-V0ApA8BQrGUAgcSc

but in this case if you do not have the cell lines and the specific intrumentation for growth (CO2 shaker incubator is required), probably you can ask the support of some company that offer protein expression service in mammalian as

biointron

https://www.biointron.com/recombinant-protein-expression/recombinant-protein-expression-in-mammalian-cells.html?_gl=1*18d9q8b*_up*MQ..*_ga*MTgzNDk2NjExNy4xNzI0OTI5OTg1*_ga_NG51YMR8QX*MTcyNDkyOTk4NC4xLjAuMTcyNDkyOTk4NC4wLjAuMjY1NTk4Nzgy

or

Genescript

https://www.genscript.com/recombinant-protein-services.html?src=pullmenu&_gl=1*8bmdz3*_up*MQ..&gclid=CjwKCAjwuMC2BhA7EiwAmJKRrDeOc8D9mkoaMPAYT0_RV9UiCmrq3v5DxkLemJPY-i99QuowFuIQtBoCbP8QAvD_BwE

good luck

Manuele

Thank you, Manuele!