20 mutations in a small protein like that may either lead to misfolding and subsequent degradation or to the accumulation of rare codons that don't match the preferred codon usage anymore. You could try to use E. coli expression strains with inactivated proteases in the first case and strains with extra copies of genes for rare tRNAs in the second.

Have you checked for codon bias? Rare codons = very low expression.

There is also a possibility that they proteins are toxic (inducible promotor usually fixes this, but not always). Are you seeing slow growth or clumping/stringy cultures?

You can also try the "more is more" strategy and start with a huge culture volume.

Good luck!

Dear Lina Maria Pena

considering the low MW the expression level is not so low, since in my past experience, small peptides show low expression in E.coli since they can be subjected to degradation. The problem is that coexpression of 2 chains in not every so simple.

Just some questions:

1) Does your chains contains cysteines?

2) I suspect that your 2 chains have to be associated theoretically, it yes, do you know if those chains are associated in the correct way? E.g single peak with correct theorerical MW in SEC chromatography?

Because if you would like to produce a 2 chain complex containing cisteines, as happened for mab probably the mammalian expression eg Expi293 where you can perform co-trasfection of 2 plasmids is preferable respect than E.coli

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

Moreover if you have no cisteines and you chains associate well in a single peak and the only problem is the productivity, you can try different approaches to improve it in E.coli

It depends a lot how much you need to improve your yields:

If not a lor, you can try to just improve the culture conditions by using a high cell density media, as enpresso for example which will allow to you to improve volumetric yields of your process

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

https://www.blogger.com/video.g?token=AD6v5dwU8Sl-43BfVTSeIRFBcQiWXAeg80Ud5UCxiXqUj4AFUO7p1BOHHmV4NiV0MY32A41mTgUH2-nyouDVBhcVFOuGc1lJ5_1WgP3RrsvakB6iJHCW_JOnrD8kIeiUSimWYahOs-OP

Otherwise you have to try more complex approaches as addition of N-terminal fusion tag as GB1, TRX, but i'm not sure that it may affect the assemply of your complex.

good luck

Manuele

Hay que ver todas las ordenaciones, calcular la transferencia de electrones de dos en dos y hacer los cálculos vectoriales para obtener resultantes, luego calcular interacciones con respecto a la sustancia fija.

Es mucho trabajo usando química cuántica.

Hi Lina!!

I want to share an interesting approach for enhancing the overexpression of recombinant proteins that I've come across. It involves the use of 3% ethanol in the culture medium, which has been shown to significantly boost protein expression levels. There’s a verified and established protocol documented in the paper titled "An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli" (PubMed ID: [26629417](https://pubmed.ncbi.nlm.nih.gov/26629417/).

This method has been cited by multiple studies, indicating its effectiveness and reliability. I encourage you to give it a try in your work, as it may prove beneficial for your recombinant protein expression endeavors. Let me know how it goes! Good Luck.