Essential myosin light chain MLC-1sa decelerates actin and thin filament gliding on beta-myosin molecules

Osten, Jennifer; Mohebbi, Maral; Wang, Tianbang; Uta, Petra; Kraft, Theresia; Amrute-Nayak, Mamta; Scholz, Tim

doi:10.1016/j.bpj.2021.11.1446

Cited by 1 publication

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“…Another possibility that is less likely but that we cannot exclude based on our available data is that reduced or different co-purification of essential or regulatory light chains (cf. [ 43 , 44 , 45 ]) contributes to the lower velocity with GenJet transfection.…”

Section: Discussionmentioning

confidence: 99%

Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent

Berg,

Velayuthan,

Månsson

et al. 2024

IJMS

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Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac β-myosin heavy chain (β-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

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Section: Discussionmentioning

confidence: 99%