1992 Fiscal Year Final Research Report Summary
Development of procedures to knock out the function of intracellular proteins by expression of cDNAs encoding specific antibodies.
| Project/Area Number |
02557013
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Tokyo University |
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Principal Investigator |
NUKADA Toshihide Tokyo University, Faculty of Medicine, Assistant Professor, 医学部(医), 講師 (80189349)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Masato Mitsubisi Kasei Institute of Life Science, Department of Biomolecular Research,, 生体分子科学研究部, 主任研究員
SATO Showbu Mitsubisi Kasei Institute of Life Science, Department of Biomolecular Research,, 生体分子科学研究部, 主任研究員
HAGA Tatsuya Tokyo University, Faculty of Medicine, Professor, 医学部, 教授 (30011646)
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| Project Period (FY) |
1990 – 1992
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| Keywords | GTP-binding protein / Monoclonal Antibody / cDNA Cloning / Immunoglobulin / Phospholipase C / Pertussis Toxin / Baculovirus / Muscarinic Acetylcholine Receptor |
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| Research Abstract |
1. cDNA cloning of two novel GTP-binding protein alpha-subunits (Galphas) from a bovine liver cDNA library. These alpha-subunits (designated as GL1alpha and GL2alpha) lack apparent ADP-ribosylation sites for pertussis toxin. 2. Expression of exogenous Galphas in neuronal cells and Xenopus oocytes. Exogenous Gi3alpha mediated muscarinic receptor- (MAchR) dependent inhibition of high-threshold, voltage-dependent Ca^<2+> currents in NG108-15 cells Exogenous GL1alpha and GL2alpha that coupled to both the m1 subtype of mAchR and metabotropic glutamate receptor 1 mediated inhibition and stimulation of phospholipase C activity in Xenopus oocytes, respectively. 3. preparation of monoclonal antibodies specific for GL1alpha, GL2alpha, Gi1alpha, Goalpha using cell fusion method. 4. cDNA cloning of the specific monoclonal antibodies for each Galpha. The primary structures of the variable regions of the immunoglobulin heavy (gamma1 and mu) and light (kappa) chains were determined from the respectiv
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e cDNAs. 5. Establishment of an E. coli system for the simultaneous expression of mu and kappa cDNAs for anti-GL1alpha and Gi1alpha monoclonal antibodies. mu and kappa proteins secreted into the periplasmic space formed disulfide bonds and may be mu_2kappa_2.6. Mass production of Galpha proteins using a baculovirus expression system. solubilization of GL1alpha and GL2alpha proteins from membrane fractions by Lubrol PX or sodium cholate was made possible by the c-expression of G-protein beta_1gamma_2-subunits. 7. Insertion of immunoglobulin heavy and light chain cDNAs into expression vectors that contain an inducible promoter derived from mouse mammary tumor virus or Drosophila heat-shock protein 70 gene. We are planning to co-transfect vectors expressing the heavy and light chains of monoclonal antibodies for specific Galphas into cultured cells. We will then study the intracellular changes that result from the inhibition of specific subtypes of Galphas following glucocorticoid- or heat- induction of monoclonal antibody synthesis. Less
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