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Cloning, expression, purification, crystallization and preliminary X‐ray diffraction analysis of recombinant human fumarase
Author(s) -
Pereira de Pádua Ricardo Augusto,
ato Maria Cristina
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x13033955
Subject(s) - fumarase , cloning (programming) , molecular cloning , gene , recombinant dna , escherichia coli , chemistry , microbiology and biotechnology , biochemistry , biology , gene expression , computer science , programming language
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.