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Bacteria (e.g E.coli)
 Expression of eukaryotic proteins in prokaryotic cells is a widely used system. E.coli is the least expensive host for production of recombinant proteins and peptides that do not require post-translational modifications.
Intracellular expression of protein in E.coli most commonly results in insoluable inclusion bodies and is often the preferred approach at industrial scale. Inclusion bodies require solubilization followed by renaturation. Often these folding and refolding pathways are product specific.
The product may also be expressed in a soluble state in the cytoplasm or secreted to the periplasmatic space. Secreation into the periplasm can be achieved by fusing the N-terminus to a signal peptide, a secretory leader, though, its application at an industrial scale is limited. Proteins can also be expressed as fusion proteins, a construct which provides an affinity handle which facilitates later purification.
Validation issues typically include demonstration of the removal of endotoxins, nucleic acids, host cell proteins and terminal methionine product variants. Incorrectly folded proteins and proteins modified by proteases are also a concern.
Advantages
- Simple and well characterized genetics
- Rapid cell growth (doubles in 20-30 min)
- Easy to grow in inexpensive culture media
- Easy to scale up fermentation
- High expression levels
Disadvantages
- Cell disruption gives more complex purification problems
- Inclusion body formation; solubilization and refolding required
- Expression of N-terminal methionine product variants
- Reducing conditions of cytoplasm may give improper folding of protein
- Lack of glycosylation and other post-translational modifications
- Presence of endotoxin and host cell proteins
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Read more about how to produce recombinant proteins with insect cells, mamalian cells, transgenics, and yeast.
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