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To answer #4...I have had success with GeneBee (http://www.genebee.msu.su/services/rna2_reduced.html) for RNA secondary structure predictions, and for codon usage analysis, http://www.faculty.ucr.edu/~mmaduro/codonusage/usage.htm. These two tools helped me fix an expression problem for our lab. Peter J. Miller Collins Laboratory Department of Biochemistry and Biophysics University of North Carolina at Chapel Hill 919-966-9410 On 1/10/07 12:10 PM, "Ailong Ke" <[EMAIL PROTECTED]> wrote: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > 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? > > > >
