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: Ricardo Augusto Pereira de Pádua, Maria Cristina Nonato
• Journal: Acta crystallographica. Section F, Structural biology communications (Acta Crystallogr F Struct Biol Commun) Vol. 70 Issue Pt 1 Pg. 120-2 (Jan 2014) ISSN: 2053-230X [Electronic] United States
• PMID: 24419633 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Recombinant Proteins
• Fumarate Hydratase

Topics

• Amino Acid Sequence
• Cloning, Molecular
• Crystallization
• Electrophoresis, Polyacrylamide Gel
• Fumarate Hydratase (chemistry, isolation & purification)
• Humans
• Molecular Sequence Data
• Recombinant Proteins (chemistry, isolation & purification)
• X-Ray Diffraction