title: Preparation and characterization of recombinant murine p65/L-plastin expressed in Escherichia coli and high-titer antibodies against the protein.
abstract: We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells. p65/L-plastin is characterized by a series of Ca(2+)-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to beta-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.
authors.affiliation: Department of Immunology and Host Defenses, Ehime University School of Medicine, Ehime 791-0295, Japan. hiroto@m.ehime-u.ac.jp
authors.forename: Hiroto
authors.initials: H
authors.lastname: Shinomiya
authors.forename: Kozo
authors.initials: K
authors.lastname: Nagai
authors.forename: Hajime
authors.initials: H
authors.lastname: Hirata
authors.forename: Naoto
authors.initials: N
authors.lastname: Kobayashi
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Hasegawa
authors.forename: Fengzhi
authors.initials: F
authors.lastname: Liu
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Sumita
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D000917:Antibody Formation; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D007150:Immunohistochemistry; D007962:Leukocytes / Q000737:chemistry; D051379:Mice; D008840:Microfilament Proteins; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000906:Antibodies; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C066559:Lcp1 protein, mouse