title: Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system.
abstract: 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 have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. 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 a 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. 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.
authors.affiliation: Department of Chemistry & Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-1030, United States.
authors.forename: James T
authors.initials: JT
authors.lastname: Caldwell
authors.forename: Girish C
authors.initials: GC
authors.lastname: Melkani
authors.forename: Tom
authors.initials: T
authors.lastname: Huxford
authors.forename: Sanford I
authors.initials: SI
authors.lastname: Bernstein
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism*; D005426:Flight, Animal; D015870:Gene Expression; D055786:Gene Knockout Techniques; D009218:Myosins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*
chemical_list: D020033:Protein Isoforms; D009218:Myosins