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Transgenic Expression and Purification of Myosin Isoforms Using the Drosophila melanogaster Indirect Flight Muscle System
Author(s) -
Caldwell James Tore,
Melkani Girish C.,
Huxford Tom,
Bernstein Sanford I.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb204
Subject(s) - myosin , drosophila melanogaster , gene isoform , biology , transgene , mutant , recombinant dna , microbiology and biotechnology , skeletal muscle , gene , myh7 , myosin light chain kinase , genetics , anatomy
We report a novel method for the generation of extremely pure transgenic myosin to carry out crystallization screening for x‐ray crystallography. We are optimizing a novel method for the use of genetically engineered Drosophila melanogaster to express 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 heavy chain isoforms. Second, that wild‐type or mutagenized versions of the gene can then be inserted into a specific location in the Drosophila genome. Third, that the Mhc 10 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. Using these advantages we devised a method for expressing and purifying two isoforms of recombinant histidine‐tagged myosin from an engineered fly strain. The myosin shows ATPase activity and is of sufficient purity and homogeneity for crystallization. This 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. Supported by NIH R01 GM032443 to SIB.