Dear Emmanuel Kabwe

it depends a lot from the conformational stability of your sequence.

Are you epitopes linear o conformational epitopes?

Did you insert the epitpopes in a structured protein scaffold of just link them using linkers? Did you use flexible (eg GSGSGS) linker or rigid linkers (e.g (EAAAK)

Did you peptides contains cisteines that can be form S-S bonds?

if you are just fuse linear epitopes with a flexible linkers i doubt that you protein will acheive a proper conformation.

best regards

Manuele

Odds are against you, most proteins expressed in E. coli are produced as inclusion bodies.

Manuele Martinelli, I really appreciate the time you got from your busy schedule to try and help me to solve this problem. Your answer, your explanations are what I need.

Im actually planing to design a multi-epitope molecule combining together different viruses of the same genus. But I am afraid that I will not be able to purify the protein with stability, properties and proper conformation.

Can this be achieved and what are the recommendations to add to the molecule so that I can get proper conformation and achieve its immunogenicity?. Im planing to conduct in vitro and in vivo experiments with this protein after its purification.

Omar Gonzalez-Ortega thank you for your answer and your time. Yes, this is a big problem but we can still solubilise and refold it as long as we are sure that this kind of protein can achieve proper conformation after purifying it.

Im really wondering if the protein designed like this can achieve proper conformation.

Dear Emmanuel Kabwe

with "diffrent viruses for the same genus" did you mean the different epitopes from the same antigen of different virus variants? as for example epitopes of different RBD variant of the covid virus?

in this case probably the best is identity the smallest folded domain of the antigen that could be successfully produced in E. coli with proper conformation and afterward desing a fusion protein containing multiple domains linked with spacers as GSGSGSGS link.

Take in consideration that ofter the virus surface proteins contaning structural S-S bonds whose are not easy to be produced in E.coli and probably the use of a mammalian expression sistems, eg EXPI293 could be preferable to express this kind of proteins.

best

Manuele

Dear Manuele Martinelli,

Sorry for the confusion, I meant for example Orthohantavirus genus then we have species like Andes, Sin Nombre, Laguna, Choclo etc. We want to create multi-epitope molecule derived from only immunogenic epitopes targeting B and T cells of these species. Now our worry is if it will be possible to get the proper conformation, which is immunogenic etc of this molecule after purification(

Thank you very much for your professional recommendations and your time.

Yours in science

Emmanuel