| RECOMBINANT PROTEIN PRODUCTION |
| Proteins are chains of amino acids and these chains must be folded in the correct way for the protein to be active. Production of proteins can fail due to difficulties in obtaining the correct 3-D structure. |
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| PROTEIN EXPRESSION |
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| Recombinant DNA technology is the favoured method of producing proteins as drugs or for research purposes. The key to this technology is the use of microbes (e.g. the bacterium E. coli) as miniature protein bio-factories. |
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| INCLUSION BODIES |
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To achieve high yields each microbial cell must make a large amount of the target protein but this often accumulates in the cell as an aggregate of inactive protein (an inclusion body).
Successful production depends on being able to release the protein, solubilise it and then refold it to the correct and active 3-D structure.
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| PROTEIN REFOLDING |
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| This refolding stage has long been — and still is — considered a “black art” and frequently leads to projects being abandoned, laborious factorial screens being employed and detracts from the inherent advantages of using high expressing, inexpensive E. coli as an expression platform. |
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| Expedeon’s NVoy Polymer technology is changing this view and improves the chances of success with a simple, generic refolding process. |
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The first step in the refolding process is to solubilise the aggregated target protein (inclusion body) with chemicals (denaturants) such as urea or guanidine hydrochloride. Traditionally the denaturants are then removed or diluted to a low level allowing the protein to refold. Unfortunately during this refolding process the proteins often collapse too quickly, preventing the correct hydrogen bonds from forming resulting in inactive or irreversibly aggregated protein.
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| PROTEIN AGGREGATION - THE PROBLEM |
| The problem is that the proteins must pass from an extended (denatured) conformation through intermediate conformations before finally reaching the correct and active structure. It is the refolding intermediates that are highly prone to aggregation and responsible for yield losses. |
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| Expedeon’s proprietary NVoy Polymer technology protects the protein during the vulnerable stages in the refolding process. |
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| PROTEIN PROTECTION: EXPEDEON’S SOLUTION TO REFOLDING |
| The structure and mode of action of the NVoy molecules makes them particularly suited to assist protein refolding. Generally, the hydrophobic amino acids of a native protein tend to be sequestered into the protein core. However, when the protein is denatured a higher number of hydrophobic amino acids are exposed to the aqueous phase. |
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| The amphipathic NVoy molecules exploit this change by binding tightly to refolding intermediates – the hydrophobic parts of the molecules associating with the exposed hydrophobic amino acids with the resulting decrease in entropy - thus preventing aggregation and then loosening their association as the protein molecules collapse and refold. |
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| SUMMARY |
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| Conventional Refolding Often Results in... |
- Researchers using expensive, often lower yielding, expression systems
- Researchers using ineffective refolding screens for each and every protein
- Projects being shelved or abandoned as refolding is not seen as a viable option
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| Our Approach is unique in that it... |
- Is Generic and works for a wide range proteins
- Requires only small amounts of protein to get an answer
- Allows you control the process - use your expert knowledge of your protein to dictate how quickly to refold the protein
- Is quick and simple to use
- It does not modify the folded protein in any way
- Is easy to remove when no longer needed
- Does not Involve the use of extreme conditions
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