Solublizing the insoluble
Biochemical and structural research on target proteins has often been hindered by the difficulties faced in protein purification as proteins are known to aggregate in unsuitable environments. Therefore, researchers have often resorted to using denaturing conditions to purify proteins and consequent...
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| Format: | Final Year Project |
| Language: | English |
| Published: |
2012
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| Online Access: | http://hdl.handle.net/10356/49249 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | Biochemical and structural research on target proteins has often been hindered by the difficulties faced in protein purification as proteins are known to aggregate in unsuitable environments. Therefore, researchers have often resorted to using denaturing conditions to purify proteins and consequent refolding. However, this has resulted in proteins with low biological activity. Protein insolubility is often due to the change in the native protein folding conformations, exposing its hydrophobic sites and forming aggregates. Hence, we decided to look into a systematic screening of buffers to optimize the conditions to increase protein yield from bacterial culture for its purification and storage. p53, being notoriously known as a “difficult” to purify protein, was chosen to be worked on. SDS-PAGE and western blot techniques were employed to compare the protein solubility between in each buffer combinations. Tris-HCl and phosphate buffers were found to be optimized for solubility of p53. However, Tris-HCl was incompatible with the downstream purification steps. To validate the protein activity, DNA interactions with the immobilized p53 was measured using surface plasmon resonance biosensor. In the process, we realized that the conditions optimized and compatible for purification were not the best conditions for storage. |
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