Graduate Thesis Or Dissertation

 

Purification and characterization of recombinant calpain-5 Public Deposited

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  • Recombinant human calpain-5 was expressed in insect cells using a baculovirus system. The expressed calpain-5 was purified by both traditional chromatography and by affinity-column chromatography. Both methods yielded active protease. Calpain-5 displayed very limited hydrophobicity. This indicated that calpain-5 is not a membrane binding protein. Calpain-5 had pI of 8.3. The recombinant calpain-5 also exhibited calcium-dependent proteolytic activity. The calculated calcium requirement for half-maximal activity was 9.6 mM when incubated at 37°C and 26.5 mM when incubated at 30°C. Compared to traditional calpains, which require less than 1 mM calcium for half-maximal activity, calpain-5 exhibited weaker proteolytic activity. This is an unusual observation because calpain-5 lacks the typical calcium-binding domain of the calpains and implied that other calcium-binding region of the protein account for calcium-binding and sensitivity. Our results also showed that calpain-5 was different from traditional calpains because its activity was higher at 37°C compared to 30°C and remained active at 37°C for more than 2 hours. This differs from traditional calpains which display better proteolytic activity at lower temperatures and become inactive within 30 minutes of incubation in 37°C. Calpain-specific inhibitors, calpastatin and E64, did not inhibit calpain-5. Only one calcium-binding inhibitor, PD150606, inhibited calpain-5 proteolytic activity. These results confirmed that calpain's calcium-binding domain is important in calpastatin binding and calpain-5 possesses other calcium-binding regions. Calpain-5 was able to degrade spectrin, a ubiquitous cytoskeletal protein. This indicates that calpain-5 might have a role in cell remodeling. Finally, calpain-5 has the ability to degrade itself. It is not clear if this is the result of inter- or intra-molecular proteolysis and whether this leads to activation of the protein or is, instead, the first step in its degradation. Calpain-5 is expressed at highest concentrations in testis, brain, liver and gastrointestinal tract. It is not clear why these tissues require a unique calpain. Calpain-5 may provide these tissues with an additional calcium-dependent proteolytic activity which is not regulated by calpastatin and which could participate in cytoskeletal protein turnover.
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