AN INVESTIGATION OF THE EFFECTS OF MUTATIONS IN THE CALPAIN RECOGNITION SITE OF CARDIAC MYOSIN BINDING PROTEIN-C ON PURITY OF RECOMBINANTLY EXPRESSED PROTEIN
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PublisherThe University of Arizona.
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AbstractCardiac myosin binding protein C (cMyBP-C) is a cardiac specific thick filament protein that is essential for the regulation of cardiac muscle contraction. Mutations in the cMyBP-C gene have been directly linked to cardiomyopathies such as hypertrophic cardiomyopathy (HCM), which is the most common form of genetic heart disease. Calpain, a calcium-dependent protease, has a recognition site in cMyBP-C that is accessible when the cMyBP-C is dephosphorylated, causing proteolysis. Previously purified recombinant cMyBP-C proteins have had an unwanted band at a size that suggests calpain digestion of the full length construct. In order to investigate the effects of mutations to the calpain recognition site of cMyBP-C on recombinantly expressed protein purity, a multistep methodology was used. Two mutant cMyBP-C plasmids were constructed that contained altered calpain sites, one including a deletion of the calpain site and the other containing a leucine to alanine substitution in the site. The wild-type (non-mutated) and calpain mutant constructs were used to generate proteins to test if these modifications altered purity of recombinant cMyBP-C protein. After bacterial expression and immobilized metal affinity column purification, the final protein yield of the substitution construct was 14.19 mg and the final protein yield of the deletion construct was 36.32 mg. However, when run on a polyacrylamide gel, both of these constructs still had the unwanted band suggesting cleavage, possibly by an unknown protease. Following this, an alternative lysis and purification protocol was used to create wild type cMyBP-C; however, this too left undesired bands prevalent in the protein. It can be concluded that an altered or even distinctly different purification methodology must be investigated in the future to successfully eliminate the unwanted degradation products in purified cMyBP-C.
Degree ProgramMolecular and Cellular Biology