Dear Samukelisiwe Mothae

i think that you can produce the N-terminal extracellular TRL4 domain in E:coli while produce the entire full lenght protein could be quite challenging since it is a transmembrane protein and the extraction and purification of trasmembrane proteins is something very challenging.

Otherwise you can try to express the fulll neght in the outer membrane vesiches of E.coli by using a E.coli strain (e.g delta tolR or delta OmpA) able to over produce the outer membrane vescicles on the culture media, but in this way you will not obtain the pure protein, but the protein expressed into those vesicles that contains other e.coli proteins.

it depends a lot from the final target of your experiments.

good luck

Manuele

Thank you so much for your assistance and answer Manuele Martinelli . I also thought the same thing that TLR4 is a transmembrane protein and maybe that might hinder it expression. Thank you very much

Dear Samukelisiwe Mothae

there are several TLR4 ECD (extracellular domains) commercially avialable, which confirms that the production of this domain is possible.

e.g

https://www.sinobiological.com/recombinant-proteins/human-tlr4-10146-h08b

https://www.novusbio.com/products/recombinant-human-tlr4-protein-cf_1478-tr

https://www.thermofisher.com/proteins/product/Human-TLR-4-Recombinant-Protein/160-06-100UG?gclid=CjwKCAjw4ri0BhAvEiwA8oo6F4aiDlPEtt61fqbtaQpuDWcqPk4vWRyxs71fMpEIkQcGJuM_OPAlQBoCEXcQAvD_BwE&ef_id=CjwKCAjw4ri0BhAvEiwA8oo6F4aiDlPEtt61fqbtaQpuDWcqPk4vWRyxs71fMpEIkQcGJuM_OPAlQBoCEXcQAvD_BwE:G:s&s_kwcid=AL!3652!3!698069621005!!!g!!!21251360860!165356725881&cid=bid_rep_pep_r01_co_cp0000_pjt0000_bid00000_0se_gaw_dy_pur_con&gad_source=1

but most them are produced in mammalian (eg HEK293) or baculovirus.

i think that you can try in E.coli , but to have some probability you have al least to couple periplasmic expression coupled with codon optimization.

However the presence of 16 cysteines in the ECD domain lead me to believe that probably to obtain good yields and folding the mammalian expression sistem as Expi293 or ExpiCHO can be the best solution.

https://www.blogger.com/video.g?token=AD6v5dyTuEwDXD2ztCnLIFHkG06cCp1jaRuUHOGFtHdiTFnj-yS_TyCyPqzvi6RogJaSP_sXqbJnMDIYplkXODpfOc21BETPPh74J7FJuwMPxHW4X79mURcAl7-V0ApA8BQrGUAgcSc

In case you would like to try to produce it in mammalian cellsl but you do not have access to cells and instrument required, Biointron (https://www.biointron.com/) and Genescript (https://www.genscript.com/recombinant-protein-services.html?src=pullmenu) are companies those provide good and quite cheap custom recombinant protein production services in mammalian.

good luck

Manuele

Thank you so much Manuele Martinelli this was really good knowledge and I have a good and clear understanding on the expression of TLR4. Thank you so much. I also think using mammalian cells are the best for expressing TLR4.