mRNA secondary structure and protein expression level

danielak at intra.niddk.nih.gov danielak at intra.niddk.nih.gov
Mon Sep 16 13:45:15 EST 2002

Thanks a lot for thinking about my problem. My mRNA secondary structure had in a region of RBS deltaG –8,7 and ATG was a part of a helix with deltaG –22.3. When I compared mRNA structure of other genes, which I expressed in the same vector and got at least 20mg/l, I did not observed such potentially “troublemaking” structure (but I am not an expert in RNA structures). Genes I am working with are bacterial ribonuclease inhibitors and this problematic one comes form Saccharopolyspora erythraea  (previously Streptomyces erytheus). We have expressed in our lab a couple of streptomyces genes in E.coli in high levels and I had to change a codon usage just once – not for streptomyces gene, but Bacillus subtilis, when the expression was improved from zero (or from a level that I could not detect any protein) to at least 10-20mg/l. 
I have to try another method for detecting my protein – currently I am just testing its activity – if it inhibits ribonuclease; with this assay I should be able to detect 5-10 ng of an active inhibitor. I am thinking about to add His tag and check if any protein is being expressed by western blot.  
This gene has been cloned using T7 phage display system, so I would guess that it might not need any very special conditions for correct folding.?


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