title: Biochemical reconstitution of the WAVE regulatory complex.
abstract: The WAVE regulatory complex (WRC) is a 400-kDa heteropentameric protein assembly that plays a central role in controlling actin cytoskeletal dynamics in many cellular processes. The WRC acts by integrating diverse cellular cues and stimulating the actin nucleating activity of the Arp2/3 complex at membranes. Biochemical and biophysical studies of the underlying mechanisms of these processes require large amounts of purified WRC. Recent success in recombinant expression, reconstitution, purification, and crystallization of the WRC has greatly advanced our understanding of the inhibition, activation, and membrane recruitment mechanisms of this complex. But many important questions remain to be answered. Here, we summarize and update the methods developed in our laboratory, which allow reliable and flexible production of tens of milligrams of recombinant WRC of crystallographic quality, sufficient for many biochemical and structural studies.
authors.affiliation: Department of Biophysics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.
authors.forename: Baoyu
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Biophysics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.
authors.forename: Shae B
authors.initials: SB
authors.lastname: Padrick
authors.affiliation: Department of Biophysics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Henry
authors.affiliation: Department of Biophysics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA. Electronic address: Michael.Rosen@utsouthwestern.edu.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Rosen
mesh_terms: D000199:Actins; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007313:Insecta / Q000166:cytology; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000199:Actins; D011994:Recombinant Proteins; D051300:Wiskott-Aldrich Syndrome Protein Family
keywords: Actin; Protein complex; Reconstitution; WASP; WAVE; WRC