title: Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of recombinant human fumarase.
abstract: Human fumarase (HsFH) is a well-known citric acid cycle enzyme and is therefore a key component in energy metabolism. Genetic studies on human patients have shown that polymorphisms in the fumarase gene are responsible for diseases such as hereditary leiomyomatosis and renal cell cancer. As a first step in unravelling the molecular basis of the mechanism of fumarase deficiency in genetic disorders, the HsFH gene was cloned in pET-28a, heterologously expressed in Escherichia coli, purified by nickel-affinity chromatography and crystallized using the vapour-diffusion technique. X-ray diffraction experiments were performed at a synchrotron source and the structure was solved at 2.1 Å resolution by molecular replacement.
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto - Universidade de São Paulo, Avenida do Café s/n, 14040-903 Ribeirão Preto-SP, Brazil.
authors.forename: Ricardo Augusto
authors.initials: RA
authors.lastname: Pereira de Pádua
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto - Universidade de São Paulo, Avenida do Café s/n, 14040-903 Ribeirão Preto-SP, Brazil.
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Nonato
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D005649:Fumarate Hydratase / Q000737:chemistry* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D014961:X-Ray Diffraction
chemical_list: D011994:Recombinant Proteins; D005649:Fumarate Hydratase
keywords: Reed's syndrome; citric acid cycle; fumarase; fumarate hydratase; leiomyomatosis; renal cell carcinoma