title: Expression, purification and crystallization of human kynurenine aminotransferase 2 exploiting a highly optimized codon set.
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 active human KAT2 suitable for future small-molecule lead compound screening and structural design work.
authors.affiliation: Group in Biomolecular Structure and Informatics, Faculty of Pharmacy, University of Sydney, NSW 2006, Australia.
authors.forename: Guanchen
authors.initials: G
authors.lastname: Sun
authors.affiliation: Group in Biomolecular Structure and Informatics, Faculty of Pharmacy, University of Sydney, NSW 2006, Australia.
authors.forename: Alireza
authors.initials: A
authors.lastname: Nematollahi
authors.affiliation: School of Molecular Bioscience, University of Sydney, NSW 2006, Australia.
authors.forename: Naveed A
authors.initials: NA
authors.lastname: Nadvi
authors.affiliation: School of Molecular Bioscience, University of Sydney, NSW 2006, Australia.
authors.forename: Ann H
authors.initials: AH
authors.lastname: Kwan
authors.affiliation: Bragg Institute, Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW 2234, Australia.
authors.forename: Cy M
authors.initials: CM
authors.lastname: Jeffries
authors.affiliation: Group in Biomolecular Structure and Informatics, Faculty of Pharmacy, University of Sydney, NSW 2006, Australia. Electronic address: bret.church@sydney.edu.au.
authors.forename: W Bret
authors.initials: WB
authors.lastname: Church
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007736:Kynurenic Acid / Q000737:chemistry / Q000378:metabolism; D007737:Kynurenine / Q000737:chemistry / Q000378:metabolism; D019636:Neurodegenerative Diseases / Q000473:pathology / Q000628:therapy*; D011487:Protein Conformation; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use; D014961:X-Ray Diffraction
chemical_list: D003062:Codon; D007737:Kynurenine; D000637:Transaminases; C021481:kynurenine-oxoglutarate transaminase; D007736:Kynurenic Acid
keywords: Codon optimization; Crystallization; Escherichia coli; HPLC; Kynurenine aminotransferase; Ni-NTA