Preparation and characterization of recombinant murine p65/L-plastin expressed in Escherichia coli and high-titer antibodies against the protein.
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.
Shinomiya
Hiroto
H
Department of Immunology and Host Defenses, Ehime University School of Medicine, Ehime 791-0295, Japan. hiroto@m.ehime-u.ac.jp
Nagai
Kozo
K
Hirata
Hajime
H
Kobayashi
Naoto
N
Hasegawa
Hitoshi
H
Liu
Fengzhi
F
Sumita
Kohsuke
K
Asano
Yoshihiro
Y
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
D000906:Antibodies; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C066559:Lcp1 protein, mouse