Purification and characterization of recombinant human macrophage colony-stimulating factor and generation of a neutralizing antibody useful for Western analysis |
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| Institution: | 1. Department of Protein Chemistry, Cetus Corporation, 1400 Fifty-Third Street, Emeryville, CA 94608, U.S.A.;2. Department of Cell Biology, Cetus Corporation, 1400 Fifty-Third Street, Emeryville, CA 94608, U.S.A.;1. Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer Ufer 1-3, 68167 Mannheim, Germany;2. Fifth Medical Department, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany;3. Red Cross Blood Service Baden-Württemberg–Hessen, Friedrich-Ebert Str. 107, D-68167 Mannheim, Germany;4. Institute of Carcinogenesis, N.N.Blokhin Russian Cancer Research Center, Moscow, Russian Federation;5. Laboratory for Translational Cellular and Molecular Biomedicine, Tomsk State University, 36 Lenin Prospekt, Tomsk 634050, Russian Federation;6. University of Groningen, University Medical Center Groningen, Dept. Pathology and Medical Biology, The Netherlands;1. Maharaja Agrasen Institute of Technology, Delhi, India;2. Sastra University, Tanjore, India;3. Graduate Program in Applied Informatics, University of Fortaleza, Fortaleza, CE, Brazil;1. School of Materials Science and Engineering, Nanjing University of Science and Technology, Nanjing, 210094, China;2. Key Laboratory of Controlled Arc Intelligent Additive Manufacturing Technology, Nanjing University of Science and Technology, Nanjing, 210094, China;3. Nanjing Institute of Technology, Nanjing, 211167, China;1. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China;2. University of Chinese Academy of Sciences, Beijing 100049, China;3. Beijing Hualishi Scientific Company Ltd., Beijing 101312, China |
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| Abstract: | Recombinant human macrophage colony-stimulating factor 1 (rCSF-1, also known as M-CSF) has been purified in milligram quantities from culture supernatants of SV40-infected CV-1 monkey cells that were transformed with a plasmid (pcCSF17) containing a human CSF-1 cDNA (Kawasaki et al. (1985) Science 230, 291–296). The rCSF-1 was purified using a 4-step procedure which resulted in a 285-fold purification and a yield of 40%. This rCSF-1 was shown to be a dimeric, disulfide-linked glycoprotein with an apparent native molecular weight of 65 kDa. The specific biological activity and amino-terminal sequence of this rCSF-1 were shown to be identical to that reported for native CSF-1 from MIA PaCa-2 cells. Although the pcCSF17 CSF-1 cDNA sequence coded for a mature polypeptide of 224 amino acids in length, C-terminal analysis of purified rCSF-1 indicated that C-terminal proteolytic processing had occurred at or near residue 158.A high-titer, polyclonal antibody to rCSF-1 was produced in rabbits and shown to specifically neutralize the biological activity of both CV-1 rCSF-1 and native CSF-1 from MIA PaCa-2 cells. In addition, the anti-CSF-1 antibody has been used to detect native and recombinant CSF-1 on Western blots. |
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