Purification of Gc-2 globulin from human serum by displacement chromatography: a model for the isolation of marker proteins identified by two-dimensional electrophoresis |
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| Authors: | A R Torres G G Krueger E A Peterson |
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| Institution: | 1. Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205, USA;2. Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut 06504, USA;3. Division of Dermatology, Department of Medicine, University of Utah, Salt Lake City, Utah 84132 USA;1. Merck & Co., Inc., Kenilworth, New Jersey 07033;2. NMR Application, Bruker BioSpin AG, Industriestrasse 26, 8117 Fällanden, Switzerland;1. Department of Pharmacokinetics and Pharmaceutical Technology, Miguel Hernandez University, San Juan de Alicante, 03550 Alicante, Spain;2. Department of Pharmacokinetics and Pharmaceutical Technology, University of Valencia, Av. de Blasco Ibáñez, 13, 46010 Valencia, Spain;3. Molecular Recognition and Technological Development, Polytechnic University −University of Valencia, Camí de Vera, s/n, 46022 Valencia, Spain;4. I3S-Instituto de Investigação e Inovação em Saúde, University of Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal;5. INEB-Instituto de Engenharia Biomédica, University of Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal;6. CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central da Gandra 1317, 4585-116 Gandra, Portugal;1. School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, PR China;2. Department of Chemistry, Innovative Drug Research Center, Shanghai University, Shanghai 200444, PR China;3. State Key Laboratory of Organometallic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, PR China;4. Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, PR China |
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| Abstract: | A protein that was initially known only as a minor spot in two-dimensional electrophoresis patterns of serum obtained from certain psoriasis patients, particularly those with a pustular component to their disease, has been purified by two stages of ion-exchange displacement chromatography on DEAE-Sephacel at different pH levels, followed by elution chromatography on hydroxylapatite. The purification was followed by examining the column fractions directly by two-dimensional gel electrophoresis. The capacity of the displacement system, which utilized carboxymethyldextrans as displacers, was very high; 6 ml of dialyzed serum applied to a 7-ml column in the initial stage resulted in a very substantial enrichment of the target protein. The second displacement stage yielded a highly purified product, contaminated only by A-1 lipoprotein. The latter was removed by hydroxylapatite chromatography. The purified protein was subsequently identified as Gc-2 globulin, a vitamin D-binding protein, by immunological procedures. The results demonstrate the effectiveness of ion-exchange displacement chromatography in focusing resolving power on the relatively narrow range of affinities represented by the target protein and its immediate neighbors in a chromatogram, as well as the applicability of the system to the isolation of a protein known only by its position in a two-dimensional electrophoretic pattern. |
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