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Hi all, Here are some off-topic questions about protein expression that I would appreciate some help with. I had a difficult time to express a 90 KDa human protein in E. coli. It was fused to the C-terminus of MBP and contains a lot of rare arginine, isoleucine and proline codons. The sequenced plasmid transforms well, but the cells have difficulty to grow in large LB media, and no overexpression were observed (I have yet to do a western). Trying BL21(DE)3RIL cells did not help. I guess I need to try the Rosetta cells for the proline codons. My questions are: 1) How big a protein can E. coli handle? Am I working at the borderline here? 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. 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. 4) Can anyone recommend an on-line server to do codon/RNA secondary structure/GCcontent optimization? 4) Can anyone recommend a reliable company for gene synthesis, with reasonable $/bp pricing?
