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I am a graduate student looking to incorporate some protein engineering into my training. I have been reading a lot about E coli lysate based systems, but they still require 3rd party additives like amino acids etc.. ($$$). I need this to be ultra cheap as this will be a side show until I can show something for my efforts. I prefer a cell free workflow because of the quick turnaround from template to protein and ease of purification. My backup plan is just to add a tag at the end of my constructs but I would prefer to avoid this.

Any suggestions would be much appreciated.

Thanks

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  • $\begingroup$ There is nothing like a cheap cell free translation system if you buy it, as these systems needed a lot of optimization in the development (I once worked for a company who developed such systems). If you want it cheap, go for a tagged protein where the tag can be cleaved. $\endgroup$ – Chris May 2 '18 at 19:33
  • $\begingroup$ What kind of scale do you need? If you need some milligrams that might already rule out cell-free expression completely. Afaik cell-free expression systems are only used where normal expression is really not an option (to incorporate non-natural amino acids, when no purification is possible, for small peptides perhaps, radiolabels, that sort of stuff). $\endgroup$ – VonBeche May 3 '18 at 15:06
  • $\begingroup$ @VonBeche Really small quantities in terms of amounts and say 100AA in terms of size. I would use this platform to try and engineer protein linker. I read that you can turn around a protein quickly and I will fail a LOT. I would want to take advantage of non-natural amino acids for smFRET to get a feel for the dynamic motion. $\endgroup$ – TheCodeNovice May 4 '18 at 1:28
  • $\begingroup$ It sounds like you want to try a lot of new things at once (although I don't know which techniques you have up and running). Also, keep in mind that building good linkers shouldn't be that hard and therefore they are scientifically not that interesting. I'd expect that a standard linker would work. If it doesn't work, scale up to a few more. Because the usual lack of secondary structure in a linker usually there's not that much to engineer. If you really want to, just try a standard one vs polyproline or something like that. $\endgroup$ – VonBeche May 7 '18 at 10:18
  • $\begingroup$ @VonBeche I was hoping to make a disordered linker that can pass information between domains bound at each end. So I am looking for something more sophisticated than just a polyG peptide sequence. $\endgroup$ – TheCodeNovice May 9 '18 at 2:13

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