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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
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The Art of Gene Expression in E. coli
