Every paper I've read on continuous CFPS deals with hundreds of microliter scale reaction volumes. The results are then extrapolated to state protein production levels of 1 to 6 mg/ml.
I am setting up an experiment to test these claims.
My reaction volume is 200 ml, using extracts from human cells (total cell count 1011). It will be an uncoupled (using mRNA coded to the specific protein) continuous transaction with regular feeding of energy source and removal of waste products. I plan to run this reaction for 24 hours.
The reaction mixture will be housed (custom built) within a dialysis membrane and be stirred continuously. This is to address the reduction of the ratio of surface area to volume that happens when larger volumes are used. The dialysis membrane set up itself will be immersed within a mixture closely mirroring it's contents.
I also am thinking of supplementing the mixture with 100% oxygen enriched water.
Do you think this is will work? Am I on a fools errand?
I think you can simply go for mammalian expression vector with pET and use mab select for purification at the end. Don't you think that will be more efficient and less exhausting ?
Thank you for your input Soham, yes that would be less exhausting, although the point of the experiment is to test the thesis that CFPS can really achieve 1 to 6 mg/ml yields of Mab's. I'm eager to know if my idea of scaling up from micro liters to hundred's of milliliters is flawed in some way.
Damayanthi Durairajanayagam Good luck!
It has been similarly attempted before with considerable success
see
Article Aglycosylated antibodies and antibody fragments produced in ...
where
" These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. "
and
Article Microscale to Manufacturing Scale-Up of Cell-Free Cytokine P...
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