Can anyone think of a potential reason why dasherGFP can be expressed just fine in a cell-free system, while cometGFP doesn't seem to work? I'm not sure if it could be a translation problem or a post-translational problem.
I've ruled out disulphide bridges being inhibited (since GFP doesn't have any), so I'm wondering if there might be some differences in post-translational modifications, or maybe that cometGFP requires some chaperones absent in the cell-free reaction mixture?
Check if there are any rare codons present in the cometGFP template
Do you work with procaryotic or Eukaryotic CFPS? and with in vitro transcribed mRNA or in coupled transcription/ translation system ?
Can you send me the DNA seq of the constructs (I will check rapidly if you have a good Kozak context, good 5'UTR... ifeukaryotic CFPS).
Thanks for the replies.
The cometGFP has been optimised for expression in E. coli, so there are no rare codons.
I'm working with a prokaryotic (E. coli) coupled transcription/translation system.
Hi,
For me 3 possibilities you have to eliminate: the mRNA is not produced, the protein is not produced, the protein is not functional.
Check the promotor for transcription of your cometGFP contruct (to be sure that is the same like DashedGFP) : T3, T7 or SP6 promotor...
You could try an independant in vitro transcription, check the mRNA integrity by agarose gel migration and add it in you coupled E.Coli CPFS.
Maybe the protein is produced but not functional. Did you check the GFP production by fluorescence, radioactive labeling or western-blot? radioactive labeling is the most sensitive method so you could see if the protein is expressed with a low or good intensity. If you observe the protein by WB or radioactivity but not fluorescence, you have a problem of folding or post-translational modification.
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