کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020204 1542320 2016 5 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Expression, purification and crystallization of human kynurenine aminotransferase 2 exploiting a highly optimized codon set
ترجمه فارسی عنوان
بیان، تصفیه و کریستالیزه کردن کینورینین آمینوترانسفراز 2 انسان از یک مجموعه کدون بهینه سازی شده بهره می گیرند
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
چکیده انگلیسی


• A schema for the heterogeneous expression of kynurenine aminotransferase 2 using E. coli.
• The single step purification using histidine tagging and Ni-NTA column is utilised.
• Codon optimisation takes advantage of biases for which evidence is good.
• The purified protein is demonstrated to be suitable for attaining crystals.
• X-ray data acquired to 1.83 Å, representing the highest resolution for a structure.

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 121, May 2016, Pages 41–45
نویسندگان
, , , , , ,