Preparation and characterization of recombinant murine p65/L-plastin expressed in Escherichia coli and high-titer antibodies against the protein.
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| 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.
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| Authors | Hiroto Shinomiya, Kozo Nagai, Hajime Hirata, Naoto Kobayashi, Hitoshi Hasegawa, Fengzhi Liu, Kohsuke Sumita, Yoshihiro Asano |
| Journal | Bioscience, biotechnology, and biochemistry (Biosci Biotechnol Biochem) Vol. 67 Issue 6 Pg. 1368-75 (Jun 2003) ISSN: 0916-8451 [Print] England |
| PMID | 12843666 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
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