Expression, purification and crystallization of human kynurenine aminotransferase 2 exploiting a highly optimized codon set

Protein Expression and Purification
Guanchen SunWBret Church

Abstract

Kynurenine aminotransferase (KAT) is a pyridoxal-5'-phosphate (PLP) dependent enzyme that catalyses kynurenine (KYN) to kynurenic acid (KYNA), a neuroactive product in the tryptophan metabolic pathway. Evidence suggests that abnormal levels of KYNA are involved in many neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease and schizophrenia. Reducing KYNA production through inhibiting kynurenine aminotransferase 2 (KAT2) would be a promising approach to understanding and treating the related neurological and mental disorders. In this study we used an optimized codon sequence to overexpress histidine-tagged human KAT2 (hKAT2) using an Escherichia coli expression system. After a single step of Ni-NTA based purification the purified protein (>95%) was confirmed to be active by an HPLC based activity assay and was crystallized using the hanging-drop vapour diffusion method. The crystal system represents a novel space group, and a complete X-ray diffraction data set was collected to 1.83 Å resolution, and higher resolution data than for any reported native human KAT2 structure. The optimised method of protein production provides a fast and reliable technique to generate large quantities of ...Continue Reading

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Citations

Mar 31, 2016·International Journal of Molecular Sciences·Alireza NematollahiW Bret Church
Jun 18, 2016·International Journal of Molecular Sciences·Alireza NematollahiW Bret Church
Mar 21, 2017·Cellular and Molecular Life Sciences : CMLS·Ping SongMing-Hui Zou
Jul 2, 2016·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Alireza NematollahiW Bret Church

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