Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays.

ABSTRACT: Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesti

SUBMITTER: Velayuthan LP 

PROVIDER: S-EPMC10008826 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature