Typical Components of an E.coli expression vector- In our previous lecture we discussed the salient feature of cloning vector of e.coli but additional structural features are essential for an expression vector.

1. Promoter- This is upstream sequence to the gene and provides the docking site for RNA polymerase.

2. Ribosome binding site- Ribosome binding site including Shine-Dalgarno sequence is the docking site for assembly of ribosome.

3. Termination site- it terminates the synthesis of m-RNA.

4. Affinity tag- The presence of affinity tag either before or after gene sequence provides a mean to purify the protein using affinity chromatography.

Promoter regulates the production of protein- The structural elements of an E.coli promoter is given in Figure 24.2. The sequence at -10 and -35 are crucial to facilitate RNA polymerase and consequently determine the strength of the promoter. The nucleotide substitution in this region severaly affects the turn over of RNA polymerase binding events or transcription initiation events. Subsquently a number of promoters are designed for over-expression of protein in E.coli using a strong or a weak promoter to suit the over-expression strategy.

Figure 24.2: A generalized promoter structure in E.coli

IPTG inducible promoter- IPTG is an synthetic analogue of lactose and it is an inducer for lac operon. The lac promoter is very widely been used to construct different expression plasmid to express protein in E.coli. The different vector contains lac promoters or its derivatives:

1. The lac promoter- example of plasmid, pUC, pGEM etc.