title: Immunoaffinity Purification of the Glycosylated Extracellular Fragment of Mouse Plexin A2 Produced in a Mammalian Expression System.
abstract: Plexins are type I membrane proteins that function as receptors for semaphorins. All of the known plexins contain a large globular domain, termed the sema domain, in the N-terminal extracellular region, which interacts with semaphorins during signal transduction. Here, we describe procedures for protein production and purification that we utilized in the crystallographic study of the mouse Plexin A2 (mPlxnA2) extracellular fragment, including the sema domain. A mutant mammalian cell line, HEK293S GnTI-, was used as an expression host for the production of a crystallizable-quality mPlxnA2 fragment, which contains several N-glycosylation sites and disulfide bonds.
authors: Graduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan. nogi@tsurumi.yokohama-cu.ac.jp.
authors: Terukazu
authors: T
authors: Nogi
authors: Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan.
authors: Emiko
authors: E
authors: Mihara
authors: Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, 700-8530, Japan.
authors: Norihisa
authors: N
authors: Yasui
authors: Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan.
authors: Junichi
authors: J
authors: Takagi
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D009419:Nerve Tissue Proteins; C492113:Plxna2 protein, mouse; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins
keywords: Crystallographic analysis; Glycosylated protein; High-density cell culture; Immunoaffinity purification; Mammalian expression system; Stable expression