distacne between rbs and start codon - (Jan/30/2008 )
Ive subcloned my protein into pET21b using the NdeI site (cutting the insert with AseI, compatible sticky ends) and as a consequence there is an additional base (A) between the ribosome binding site and the start codon. I was wondering if this will lower expression levels? Im trying to express the protein in BL21(DE3) pLysS and am getting very low yields (pretty faint bands on western blot, anti-his) and cannot see it on Coomassie stained gel. As a function of time the expression levels do not increase and its also very weird but the cells do not slow down growth when I induce. Ive tried many different conditions (IPTG conc, increasing expression temp to try and force the protein into inclusion bodies) but nothing increases my yield. Any advice would be helpful. I also checked the plasmid on a gel and sequenced it completely and it is in frame with the start codon.
Where did the gene come from? It might have a bad mismatch to E. coli codon usage. Other possibilities include strong mRNA secondary structure. i don't think a single base change in RBS location will make much difference.
Actually the gene is from E. coli.
Anyone?
BL21(DE3) pLysS strain produce T7 lysozyme to reduce the basal expression of your interested protein in case your protein is toxic to the bacteria. If you are sure your interested protein is not toxic to the bacteria, you should try BL21(DE3) strain.
Alternatively you could try pET-32a expression vector. With this expression vector, I constantly got ~60-80mg fusion protein from 1 L culture, resulting in 6-9 mg pure peptide of my interested (39 Aa, cleaved from fusion protein and purified by HPLC.).
BL21(DE3) pLysS strain produce T7 lysozyme to reduce the basal expression of your interested protein in case your protein is toxic to the bacteria. If you are sure your interested protein is not toxic to the bacteria, you should try BL21(DE3) strain.
Alternatively you could try pET-32a expression vector. With this expression vector, I constantly got ~60-80mg fusion protein from 1 L culture, resulting in 6-9 mg pure peptide of my interested (39 Aa, cleaved from fusion protein and purified by HPLC.).
Im not sure if the protein is toxic or not and in fact it is possible it could be. It is a protease domain from a lager protein. Its a serine protease that is pretty non-specific. A friend of mine suggested using Rosetta (DE3) in case the protein is toxic. Do you have any thoughts on this? What would using pET32a change over pET21b?
Im not sure if the protein is toxic or not and in fact it is possible it could be. It is a protease domain from a lager protein. Its a serine protease that is pretty non-specific. A friend of mine suggested using Rosetta (DE3) in case the protein is toxic. Do you have any thoughts on this? What would using pET32a change over pET21b?
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Since IPTG did not slow down the growth of your bacteria, I think maybe you interested protein is not toxic. Or maybe your IPTG is bad already.
pET-21b places a T7 tag at the N terminus of your protein and potentially a His tage at C. pET-32a system produces a thioredoxin fused protein with His tag at N and potential His tag at C. Thioredoxin helps the solubility of the interested protein and can be cut off from the interested protein during post-expression purification (there is a thrombin cleavage site between thioredoxin and the interested protein, or you can add a thrombin cleavage site right at the N-terminal of your protein yourself.).