UC San Diego

Variation in myosin heavy chain generates muscle fiber functional diversity. The results of 30 years of experimental investigation by the Bernstein lab and others provides evidence that supports many novel nuances in the mechanochemical mechanism of myosin due to the alternative exon splicing and mutations of the myosin gene in Drosophila melanogaster. Their in vivo and in vitro studies have characterized many functional differences. Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture has largely met with failure, although high-level expression in muscle cell culture has recently been achieved at significant expense (Resnicow, Deacon et al. 2010). We have developed a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms (Caldwell, Melkani et al. 2012). This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. The X-ray structure of the first insect muscle myosin is solved using this method. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization