hi

In my opinion, the eukaryote system is better suited for this task, because in addition to the volume of production, the antibody activity is more important than its production.However, you can use the following:

1- pET22b(+) vector < recombinant protein Release in periplasmic space but expression level Relatively low.

2- E.coli strains Origamy(DE3) or Rosetta-gami (DE3)

3- for optimal expression you need codon optimization and gene synthesis and then cloning in pET vectors < expression induced by IPTG >

finally antibody affinity and activity It is much more important than the production of recombinant antibodies.

https://www.helmholtz-muenchen.de/fileadmin/PEPF/pET_vectors/pET-22b_map.pdf

good luck

Dear Somayeeh

which antibody fragments di you refer? fab ? scfv?

for fab as reza suggests probably a mamnalian expression sistem as the expi293 of thermo able to secrete it in the cell surnatant ita more performant ( you can produce.mgs from an 30 ml flask). it is expensive but really powerfull.

for scfv that are small and easier to be secreted you can try e.coli ( citoplamic with rosetta gami or shufflet7 strains or periplamic addind pelb or ompa leader peptides.

in the past i performed periplamic approaches for many scfv. it works but you need to optimize a lot the expression protocoll and the yields where quite low.

at the end for me thr best protocoll where expression in ygr media ( find composition on my blog ( https://proteocool.blogspot.com proteocool n5 page 2) using 24h induction at 17C with 1mM iptg after on growth at 30C but the yields Alsi using an high cell dendity media were quite low: eg 1-10mg/l

good luck

manuele

Manuele Martinelli

thanks for your answer

my fragment is scfv.

But I want to know that which method has better efficiency yield? and high affinity and activity?

SIncerelly i never tried the citoplasmic expression by Origami.

I was involved in optimization of production of undreds Scfv produced from a B cell repertoire screening project and in this context we found that periplasmic expression using OmpA leader in pet vector performing expression at low temperatures in the medium that i told you and Scfv purification by imac after extraction with Celllytic express (sigmal) produced Scfv that show good ability to bind the antigen when screend at the Gyrolab platform.

https://www.gyrosproteintechnologies.com/gyrolab-immunoassay-solutions

So i think that the protocol was able to produce good quality Scfv but the yiels in terms of mg was very poor. We were able to obtain an amount to test at Gyrolab from 8ml of culture in YGR media but to produce mgs of it we had to scale-up in high volumes that means

Of course the affinity was different due to the different intrinsic charatteristic of each scfv, the problems in terms of cell LYsis and so on.

However depends which is your aim. If you aiming to produce ugs for immunoassays it is ok, if you would like to produce mgs for cristallizzation trilas, you have to spend time in prodocool optimization, because E.coli is not the best strain for S-S production.

It is reported that Brevibacillus in also able to produce nice ScfArticle Expression and Characterization of a Single-Chain Variable F...

i used this host for expression and purification of clostidium toxA catalitic fragment and I like a lot it (it is easly to handle) but i never tried to use it for Scfv production, but it could represent also a possible alternative to mammalian in case your yielad in E.coli are not enough.

good luck

Manuele

Thank you very much for all your suggestions, they were very helpful.