PDF(Pubmed)
Abstract：
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.
摘要：
用于研究分离蛋白质的横纹肌肌球蛋白II的功能性肌球蛋白重链（MHC）的产生需要成熟的肌肉（例如，C2C12)用于表达的细胞。这对于分子机制的基础研究和由于MHC基因（MYH7）突变引起的心肌病等有害疾病的研究都很重要。一般来说,腺病毒载体用于转染，但是最近我们证明了通过非病毒聚合物试剂进行转染，JetPrime.由于JetPrime的成本相当高，以及无病毒表达方法的可持续性，获得一种以上的转染试剂是重要的。这里,因此，我们评估这样的候选物质，GenJet.使用人心脏β-肌球蛋白重链（β-MHC）作为模型系统，我们发现C2C12细胞的有效转染显示出与JetPrime试剂几乎一样好的转染效率。这是按照为JetPrime开发的方案实现的，因为制造商推荐的GenJet转染悬浮细胞的应用方案表现不佳。我们证明,使用体外运动试验和单分子ATP周转试验，从GenJet试剂转染的细胞中表达和纯化的蛋白质是有功能的。达到的纯化产率略低于基于JetPrime的纯化，但是它们是以明显更低的成本实现的。我们的结果表明，一种以上的基于聚合物的转染试剂可以产生有用量的活性MHC，从而证明了无病毒方法的可持续性。特别是,我们建议GenJet,由于其目前的成本降低了约4倍，对于需要更大量的给定MHC变体的应用是有用的。
