title: Expression and purification of the natively disordered and redox sensitive metal binding regions of Mycobacterium tuberculosis protein kinase G.
abstract: Mycobacterium tuberculosis protein kinase G (PknG) is secreted into host macrophages to block lysosomal degradation. The catalytic domain (∼147-405) is C-terminally flanked by a tetratricopeptide repeat domain (TPRD). The preceding rubredoxin-like metal-binding motif (RD, ∼74-147) mediates PknG redox regulation. The N-terminal ∼75 residues were predicted to show no regulatory secondary structure (NORS) and harbor the only site (T63) phosphorylated in vivo. Deletions or mutations in the NORS or the redox-sensitive RD significantly decrease the survival function. Here, we show that the RD appears only to be present in the folded, metal-bound state if ZnCl2 is added upon induction of protein expression in minimal medium. Since factor Xa cleaves at the end of its recognition site (IEGR), a modified expression plasmid for PknG1-147 was obtained by mutating the N-terminal thrombin to a factor Xa recognition site. This allows preparing PknG1-147 with its native N-terminus. We further present a fast approach to generate expression plasmids for only the NORS or the RD by site-directed mutagenesis of the expression plasmid for His-tagged PknG1-147. An expression plasmid for PknG1-75 was obtained by introducing a stop codon at position 76 and one for PknG74-174 by introducing a factor Xa recognition site before position 74. SDS-PAGE analysis shows that all fragments are highly expressed in E. coli and can be purified to high purity. Thereby, the established preparation protocols pave the route for the NMR structural characterization of PknG regulation by its N-terminal regions, which is demonstrated by the recorded initial (1)H-(15)N-HSQC spectra.
authors.affiliation: Technische Universität München, Department of Chemistry, Biomolecular NMR Spectroscopy, Lichtenbergstr. 4, 85747 Garching, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Wittwer
authors.affiliation: Technische Universität München, Department of Chemistry, Biomolecular NMR Spectroscopy, Lichtenbergstr. 4, 85747 Garching, Germany; Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. Electronic address: sonja.dames@tum.de.
authors.forename: Sonja A
authors.initials: SA
authors.lastname: Dames
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017869:Cyclic GMP-Dependent Protein Kinases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D017869:Cyclic GMP-Dependent Protein Kinases
keywords: Isotope labeling; Mycobacterium tuberculosis protein kinase G; NMR spectroscopy; Protein purification; Redox-sensitive metal binding motif; Site-directed mutagenesis