A Method for the Transgenic Expression and Purification of Skeletal Muscle Myosin II Isoforms using Drosophila Melanogaster

Abstract

There are many biologically interesting myosin isoforms that are expressed at levels too low for direct purification from in vivo sources. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We are optimizing a novel method for the use of Drosophila melanogaster that has been genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method relies on several facts. First, that the Drosophila genome contains only one gene encoding all skeletal muscle myosin isoforms. Second, that this DNA can be manipulated with molecular biology techniques and then be inserted into a specific location in the Drosophila genome. Third, that the Mhc10 fly line has endogenous myosin knocked out in the indirect flight muscles. Fourth, that the Actin88F promoter facilitates high-level expression of accessible myosin in the thoracic indirect flight muscles. Finally, that cost of fruit fly colony propagation is relatively low. Employing these advantages, we have created a system for the production of extremely pure skeletal muscle myosin. We demonstrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. This myosin shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. Our technique may prove useful for the expression and isolation of mutant myosins linked with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.