The Art of Gene Expression in E. coli



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Gene expression in
E. coli




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Tilman Maier og Lars Jessen
 1. Factors that influence Gene Expression
2. Expression vectors
3. Folding
4. Translocation
5. Protein stability
6. How to address the hopeless case
7. Safety
8. Links

1. Factors that influence Gene Expression

  • Codon Usage: there is little tRNA made for rare codons. In E. coli these are mainly AGA and AGG (Arg codons). The genes encoding the tRNAs can be co-overexpressed.

  • Promoters used should be very tight. Tight promoters are for example arabinose (BAD), rhamnose and lactose (provided that lacIQ is co-overexpressed). Note that tryptone contains sugars such as lactose that will induce expression and thus counteract your efforts to keep expression levels down under non-inducing conditions. Replacing tryptone by NZ amine A will solve this problem.

  • Translation initiation signal (Shine-Delgarno Sequence - serves to align the ribosome on the message in the proper reading frame.) Optimal: AGG AGG, the last G should be 9 bases upstream from the A of the AUG (start of translation) Start codons on mRNA are: AUG (90%, codon for Met), GUG (9%), UUG (1%) and CUG (0.1%) Avoid secondary structure involving SD sequence and the initiator AUG In polycistronic mRNAs, the initiation site should be close to the termination codon of the upstream gene.

  • Regulation of translation: is sometimes (not often) affected by sequences in the coding region: +5 and +10 should be A or T

  • RNA Stability can be increased if stem-loop structures are cloned at the 3' end of the coding region