A simple and efficient method for generating high-quality recombinant Mical enzyme for in vitro assays

Heng Wu, Ruei Jiun Hung, Jonathan R. Terman

Research output: Contribution to journalArticle

7 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)116-124
Number of pages9
JournalProtein Expression and Purification
Volume127
DOIs
StatePublished - Nov 1 2016

Keywords

  • Axon guidance
  • F-actin disassembly
  • Flavoprotein monooxygenase
  • Guidance cues
  • MICALs
  • Plexin
  • Repulsion
  • Semaphorin

ASJC Scopus subject areas

  • Biotechnology

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