I have a problem in expression of insect cytochrome P450 gene present in pQE30Xa vector placed in M15 cells. I confirmed the sequence that there is no intermediate stop codon, but still I am unable to see the expressed protein after 4hrs of various concentrations (1 mM & 2 mM) IPTG induction. Induction was performed once after reaching the bacterial culture 0.6-0.7 O.D.
Things to look for:
- Is the expression experiment working (i.e. reagents, strain, is everything OK?). You need a positive expression control, i.e. some other gene in the same vector that you know is expressed well.
- Look around in the literature. Has that protein been expressed in E. coli before? You might have to adjust your expectations.
- Absence of stop codons is not the only prerequisite for protein expression. Does your cloning strategy ensures that the coding sequence is placed in frame with an initiator Met codon, and has a properly spaced RBS?
- If an antibody is available, check expression by Western blotting. Is not the same thing to have no expression at all (indicating there's something wrong with the construction itself) than low expression, which may be solved by changing your experimental conditions.
- Vectors similar to pQE30 have been shown to undergo promoter deletions whenever basal expression is not repressed (see e.g. http://www.microbialcellfactories.com/content/8/1/8). Add glucose (2-0.2%, it does not matter) to the plates and culture media you use for preparing stocks, do not, ever, store your recombinant strains in agar plates in the fridge (and if you do, don't ever do serial passages to 'refresh' strains stored in agar plates).
- mRNA structure close to the RBS is important for translational initiation. Perhaps your CDS/promoter/5' UTR combination is not optimal. Analyze your construction using something like the RBS calculator (http://www.voigtlab.ucsf.edu/software/).
hope it helps
Thank you for providing relevant information. I will consider your suggestions and try for expression.
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