title: A simple and efficient method for generating high-quality recombinant Mical enzyme for in vitro assays.
abstract: We have recently identified a new family of multidomain oxidoreductase (redox) enzymes, the MICALs, that directly regulate the actin cytoskeletal elements necessary for the morphology, motility, and trajectory of cells. Our genetic assays reveal that Mical is both necessary and sufficient for actin organization and cellular effects in vivo and our biochemical assays with purified Mical protein reveal that Mical utilizes its redox activity to directly disassemble actin filaments. These results identify Mical proteins as novel actin disassembly factors and uncover a redox signaling mechanism that directly regulates the actin cytoskeleton. These results have also set the stage for in-depth characterization of the Mical enzyme. However, it has been difficult to obtain sufficient amounts of highly-pure Mical protein to conduct further biochemical, structural, imaging, catalytic, and other high-precision studies. Herein, we describe a means for expressing high levels of soluble recombinant Mical protein in bacteria. Likewise, we have designed a new purification strategy that enables the rapid and efficient purification of milligram quantities of highly-pure and >99% active Mical protein. This new strategy for generating large amounts of highly-pure and active Mical protein will aid research objectives designed to characterize the biochemical, enzymology, and structural biology of Mical and its effects on actin filament dynamics.
authors.affiliation: Departments of Neuroscience and Pharmacology and Neuroscience Graduate Program, Harold C. Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
authors.forename: Heng
authors.initials: H
authors.lastname: Wu
authors.affiliation: Departments of Neuroscience and Pharmacology and Neuroscience Graduate Program, Harold C. Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
authors.forename: Ruei-Jiun
authors.initials: RJ
authors.lastname: Hung
authors.affiliation: Departments of Neuroscience and Pharmacology and Neuroscience Graduate Program, Harold C. Simmons Comprehensive Cancer Center, The University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA. Electronic address: jonathan.terman@utsouthwestern.edu.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Terman
mesh_terms: D000818:Animals; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004331:Drosophila melanogaster; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004268:DNA-Binding Proteins; C496247:MICAL protein, Drosophila; D011994:Recombinant Proteins
keywords: Axon guidance; F-actin disassembly; Flavoprotein monooxygenase; Guidance cues; MICALs; Plexin; Repulsion; Semaphorin