Hi everyone,
Recently I am helping someone to obtain 2 proteins (both are glycohydrolases) from a Hydra species by using Expi293 suspension cells via secretion. He had tried once and they'd become inclusion bodies through E. coli; therefore, we are working on this.
Instead of generating proteins, cells were dying gradually after transfection. At the time of harvest, we got both completely opaque cultures. Since the genetic distances from cnidarians, either to procaryotes or to mammals, are absurdly far, the success rates of getting properly-folded proteins are somewhat of a mystery. For instance, GFP originally from jellyfish is a widespread tool for various purposes.
This time I have to examine the cell viability, but in 2 weeks, since I need to unfreeze the cells with a small passage number afresh and prepare sufficient materials to start with. Also, he'll take some to check the expressions in every part of the culture. If it's not working, what are alternatives? Like using adherent type instead, or another model systems?
Our vector is pEXPR-IBA42.
Thank you for having great patience with the question.
Hello! Maybe this Lexsy system will help to produce properly-folded protein.
https://www.jenabioscience.com/lexsy-expression
Hi,
we observed something similar with the expression of a zebrafish enzyme in mammalian cells, which we explained with different posttranslational modification patterns. I would imagine that the problem is similar (or likely even worse) with cnidarian proteins, so maybe check if there is information available for your protein on this matter. We could dramatically improve expression (or specific enzymatic activity, respectively) by using a homologous cell line also originating from zebrafish. Maybe take a look if there are any cnidarian cell lines available (ideally including transfection protocols, which would save you a lot of tinkering and optimization), if your problem is indeed modification, this may improve expression.
Alternatively, if your problem is indeed only misfolding, you may be able to refold your overexpressed protein from E. coli inclusion bodies in vitro, e.g. by high urea concentrations to regain at least some amount of activity. There are several exemplary protocols and even ready-to-use kits available for this aproach.
Kind regards
Hi Anastasia and Patrick,
Even though i didn't know the LEXSY system and its substantial knowledge, it looks so promising. I'll discuss it with him.
We thought that it is very time-consuming to follow the denaturation strategy. Therefore we decided to use eucaryotes. Meanwhile, he doubted the presence of endotoxins as well. Those still are "variables" for us. For Cnidarian cell lines, I can try to find them out.
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