A simple and rapid preparation of smooth muscle myosin 2 for the electron microscopic analysis

Springer Science and Business Media LLC
Anahita Vispi Bharda1, Hyun Suk Jung1
1Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea

Tóm tắt

AbstractThere has been an increase in the demand for purified protein as a result of recent developments in the structural biology of myosin 2. Although promising, current practices in myosin purification are usually time-consuming and cumbersome. The reported increased actin to myosin ratio in smooth muscles adds to the complexity of the purification process. Present study outlines a streamlined approach to isolate smooth muscle myosin 2 molecules from actomyosin suspension of chicken gizzard tissues. The procedure entails treating actomyosin for a brief period with actin-binding peptide phalloidin, followed by co-sedimentation and short column size exclusion chromatography. Typical myosin molecule with heavy and light chains and approximately 95% purity was examined using gel electrophoresis. Negative staining electron microscopy and image processing showed intact 10S myosin 2 molecules, proving that phalloidin is effective at eliminating majority of actin in the form of F-actin without dramatic alteration in the structure of myosin. The entire purification discussed here can be completed in a few hours, and further analysis can be done the same day. Thus, by offering quick and fresh supplies of native myosin molecules suited for structural research, specially cryo-electron microscopy, this innovative approach can be adapted to get around the drawbacks of time-intensive myosin purifying processes.

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