JoVE Journal

Biochemistry

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MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

Transcript

We're trying to answer why mutations in myosin motors cause human disease and what roles these proteins play in the hearing process. Our research focuses on understanding the molecular mechanisms of Myosin-7a and how it works with Usher proteins to assemble and maintain the cellular machinery required for hearing. Understanding the structural and functional mechanisms of Myosin-7a requires high-quality intact protein, which has been a longstanding challenge in the field.

Recently, we made significant progress and successfully purified the full-length Myosin-7a hollow enzyme, allowing for further investigation of this important protein. With this protein now available, we can investigate key features of this motor including its structure, force generation and its motility. These insights are crucial for understanding the mechanisms of Myosin-7a in hearing and the molecular defects that lead to deafness in human patients.

In the future, our lab will leverage our expertise in the multi vac system to recombinantly produce large biomolecular complexes, for example the Usher 1 complex, which is critical for human sensory functions. This approach will enable us to determine their structures and the principles underlying their roles in sensory cells.

This protocol details the procedures for recombinantly producing the human myosin-7a holoenzyme using the MultiBac Baculovirus system and for studying its motility using a tailored in vitro filament gliding assay.

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