title: Improvement of the expression and purification of Mycobacterium tuberculosis arylamine N-acetyltransferase (TBNAT) a potential target for novel anti-tubercular agents.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX13QT, UK.
authors.forename: Areej
authors.initials: A
authors.lastname: Abuhammad
authors.forename: Nathan
authors.initials: N
authors.lastname: Lack
authors.forename: Judith
authors.initials: J
authors.lastname: Schweichler
authors.forename: David
authors.initials: D
authors.lastname: Staunton
authors.forename: Robert B
authors.initials: RB
authors.lastname: Sim
authors.forename: Edith
authors.initials: E
authors.lastname: Sim
mesh_terms: D001191:Arylamine N-Acetyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002021:Buffers; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D002021:Buffers; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D001191:Arylamine N-Acetyltransferase