Dear Sanaz

which antibody format are you expressing (mab, fab, scfv)?

which leader peptide and expression promoter are you using?

which expression media and condition?

to my knowledge using an high cell density expression media as enpresso or ygr media and low induction temperaturęs ( as 20C for 24h) you can improve the yields of fab and scfv about 5 times respect lb media but however the yields are quite low. For fab and mab you can obtain good yields moving to mammalian cell as expi293 but it are quite expensive and require dedicated instrumentation as a co2 shaker incubator which is not ever available in the labs.

you can find more information about enpresso and ygr media on my blog ProteoCool https://proteocool.blogspot.com

and an example of the pratical application of it on

https://www.fasebj.org/doi/10.1096/fj.201900374R

where HTMC is the YGR reported in the blog with-out glucose.

good luck

Manuele

I would definitely try glycine and sucrose (not necessarily theTriton X-100): Article An efficient protocol to enhance the extracellular productio...

The Triton X-100 is there for the release into the medium.

Some fusion protein. Ubiquitin or thioredoxin.

And a test a set of different leader sequences.

E. coli is not the right system to do this. Why did you choose E. coli? I'm rather impressed that you got it to work at all in this inadequate host. Normally you would use hybridoma culture or ascites to make your first few R&D grade batches. Optionally move into cho later.

Dear Sanaz,

John is right. Please use different system to increase yield. CHO and other mammalian lines will produce glycosylation at different sites. The antibody will be heterogeneous and unsuitable for FDA approval. To overcome it, you have to select a single clone with a given glycosylation pattern. The simplest way to do it is by serial dilution and methods were described at least 20 years ago.

E. coli will avoid the problem of glycosylation because it simply lacks the machinery of higher systems. Scaling up E. coli production by using a bioreactor will lead to antibody leaking into the medium more than being retained in the PS.

Best regards,

Wes

Dear Peter

thanks for the respond.the info was helpful Peter Kysel

Dear Manuele

id appreciate your answer .you are right I forgot to add some details to my question .I am working with 2XYT media , T7 , HB2151 strain , and trying to express SCFV Manuele Martinelli

Dear John and Wieslaw

thanks a bunch for helping me out .Of course the Ecoli is not the best host . I was wondering if you have more information to share with me

Wieslaw Swietnicki John Schloendorn

Dear Sanaz

i think you may also try a different e.coli strains as bl21(de3) because i never used the HB2151 and probably i'm wrong but it seens to me that this cells that are ara- are good for use with plasmids carryng the pbad promoter but do not carry the t7 polimerases and in principle they are not able to work with t7 promoter.

ciao

Manuele

thanks again for sharing extra details Manuele Martinelli

Thank you for your appreciation. Nice to see the years of sieving through literature bringing fruit for somebody.

I am curious. After geting the pelet of E. coli culture, do you use some special protocol for the release of periplasmic content without disturbing the inner membrane?

I have more suggestions:

1.I guess that the periplasm will be pretty crowded with your SCFV. Some of the SCFV could be precipitated but still functional.

Something from this set could help to bring them back into solution: Article Stabilizing Additives Added during Cell Lysis Aid in the Sol...

2. Addition of redox compound glutathione (GSH), arginine or ethanol is also worth trying:

Article DsbA and DsbC-catalyzed Oxidative Folding of Proteins with C...

&

https://aem.asm.org/content/67/9/3994.full

Dear Peter

thanks a bunch for the respond .yes we are having a defined protocol but What I am trying to figure out is to improve the yield in bacteria level .But still looking forward to every possible way to do so Peter Kysel

Dear Sanaz,

You may simply add a plasmid carrying T7 promoter which would be compatible with your vector. The ara- feature could be used to control expression of T7 polymerase in your original strain.

The T7-based system could make much more protein under T7 promoter and getting it to fold your sc antibodies correctly in the periplasm may be a challenge. Switching to a cytoplasmic system would require removal of the pelB sequence and re-cloning the construct. In theory, you could have much more protein that way. I would try pET and RSF vectors to start with as they are high copy number. The pCDF and pACYC vectors are low copy number and you may simply run into low yields as before.

Wes

Dear Wieslaw Swietnicki

The detail you shared with me was so informative .I appreciate you Wieslaw Swietnicki