Abuhammad Areej, Lack Nathan, Schweichler Judith, Staunton David, Sim Robert B, Sim Edith
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX13QT, UK.
Protein Expr Purif. 2011 Dec;80(2):246-52. doi: 10.1016/j.pep.2011.06.021. Epub 2011 Jul 13.
Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) has been proposed as a drug target for latent tuberculosis treatment. The enzyme is essential for the survival of the mycobacterium in macrophages. However, TBNAT has been very difficult to generate as a soluble protein. In this work we describe production of soluble recombinant TBNAT at a reasonable yield achieved by subcloning the tbnat gene with a purification His-tag into the pVLT31 plasmid, and subsequent optimisation of the induction conditions. The expression system results in soluble protein optimised upon extended (60 h) low level isopropyl β-D-1-thiogalactopyranoside level induction (100 μM) at a temperature of 15 °C. The level of TBNAT expression obtained in E. coli has been significantly improved from ∼2 mg to a final yield of up to 16 mg per litre of culture at a purity level suitable for structural studies. The molecular mass of 31310 Da was confirmed using mass spectroscopy and the oligomerisation state was determined. The stability of TBNAT in different buffer systems was investigated by thermal shift assays and sufficient protein is now available for the screening of chemical libraries for inhibitors.
结核分枝杆菌芳胺 N - 乙酰基转移酶（TBNAT）已被提议作为潜伏性结核病治疗的药物靶点。该酶对于分枝杆菌在巨噬细胞中的存活至关重要。然而，TBNAT 作为可溶性蛋白一直很难产生。在这项工作中，我们描述了通过将带有纯化 His 标签的 tbnat 基因亚克隆到 pVLT31 质粒中，并随后优化诱导条件，以合理产量生产可溶性重组 TBNAT 的方法。该表达系统在 15°C 温度下，通过延长（60 小时）低水平异丙基 β - D - 1 - 硫代半乳糖苷（100 μM）诱导，可产生优化的可溶性蛋白。在大肠杆菌中获得的 TBNAT 表达水平已从约 2 mg 显著提高到每升培养物最终产量高达 16 mg，纯度适合进行结构研究。使用质谱法确认了其 31310 Da 的分子量，并确定了寡聚化状态。通过热迁移分析研究了 TBNAT 在不同缓冲系统中的稳定性，现在有足够的蛋白质可用于筛选化学文库中的抑制剂。
