Cloning systems designed for gene expression:
Bacterial cells are used as hosts for recombinant expression vectors designed for the production of large amounts of a recombinant protein (fusion proteins or tagged proteins).
Problems with overexpression in bacteria include toxicity of large amounts of the recombinant protein, lack of posttranslational processing, inability to synthesize very large mammalian proteins, and protein folding and solubility.
To solve the above mentioned problems:
- The use of pET-3 bacterial vectors containing T7 promoter in combination with host cells carrying the gene for T7 RNA polymersae expressed under the control of the lacZ promoter (i.e. inducible by IPTG).
- The vector is designed so that the recombinant protein is fused to an endogenous protein (fusion proteins).
- Use an affinity tag so that the recombinant fusion protein be purified by affinity chromatography. Two affinity tagging systems are GST-glutathione affinity and polyhistidine-nickel ion affinity.
