Purification, characterization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds
| Autor: | Benildo Sousa Cavada, Celso Shiniti Nagano, Plínio Delatorre, Emmanuel P. Souza, Luis A. G. Souza, Walter Filgueira de Azevedo, Raquel G. Benevides, Alexandre Holanda Sampaio, Bruno A.M. Rocha, Frederico Bruno Mendes Batista Moreno, Henri Debray, Emmanuel Silva Marinho, Joane K. Rustiguel |
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| Rok vydání: | 2008 |
| Předmět: |
Molecular Sequence Data
Bioengineering Lactose Tandem mass spectrometry Crystallography X-Ray Applied Microbiology and Biotechnology Biochemistry Chromatography Affinity chemistry.chemical_compound Affinity chromatography Sequence Analysis Protein Tandem Mass Spectrometry medicine Animals Humans Amino Acid Sequence Molecular Biology Phylogeny Chromatography biology Hemagglutination Protein primary structure Lectin Fabaceae General Medicine Carbohydrate Trypsin chemistry Seeds biology.protein Electrophoresis Polyacrylamide Gel Rabbits Plant Lectins Protein crystallization Crystallization Peptides Ethylene glycol Sequence Alignment Biotechnology medicine.drug |
| Zdroj: | Applied biochemistry and biotechnology. 152(3) |
| ISSN: | 1559-0291 |
| Popis: | The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 microg mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A. |
| Databáze: | OpenAIRE |
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