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Hello, > 1) How big a protein can E. coli handle? Am I working at the borderline > here? No, not even close. 90+42 = 132 kDa total fusion size - we've expressed much larger things (220 kDa is starting to push it a bit). > 2) I understand that rare codons cause premature termination and > degradation by tmRNA-mediated mechanism. But in reality, do I expect a > ladder of degradation bands or complete degradation? It is puzzling to me > not to see any MBP bands. The MBP vector expresses well. This is likely indicative of toxicity. You mentioned that your cells have trouble growing - another indicator. You do not specify your choice of promoter, origin of replication, etc. - these things all matter a lot. > 3) One can fix the rare codon problem by providing a rare-codon tRNA > plasmid, or ordering a synthetic gene. Are there cases where the former > approach failed, but the latter one succeeded. Not sure since we don't usually go back and check. Hopefully someone else did. > 4) Can anyone recommend an on-line server to do codon/RNA secondary > structure/GCcontent optimization? Most of the synthetic DNA companies will do that - some will want a deposit that they refund upon accepting the job to synthesize the gene. Please note that it's not just the abundance of rare codons that matters - even more important is the distribution and clustering - two rare codons for the same AA one after the other can majorly screw up expression, three in a row usually abolish it completely. > 4) Can anyone recommend a reliable company for gene synthesis, with > reasonable $/bp pricing? Depends on what you consider reasonable. I've seen prices as low as $1/bp but they were a bit unreliable. If you have the option to use them, I highly recommend either Blue Sky or DNA 2.0. Typically cost per bp goes up with the size of the ORF or dna fragment synthesized. Please note that I have no affiliation with either company beyond being a customer. If you are able to share further details on your problem, I may be able to help you in a more systematic manner :) Artem
