The facl6 (Rv1206) ORF corresponding to the protein shown in Fig. 1 was PCRamplified from the genomic DNA of Mtb H37Rv using Pfu Turbo HotStart DNA polymerase (Agilent Genomics, CA) and cloned into pET200 D-TOPO expression vector (Life Technologies, NY) for expression of N-terminal histidine-tagged FACL6 in Escherichia coli BL21 Star (DE3) cells. Sequence integrity was verified by DNA sequencing. Protein expression was induced for 4 h at 37 by adding IPTG (isopropyl-b-D-thiogalactopyranoside) to a ultimate concentration of 1 mM when the culture achieved an optical density at 600 nm (OD600) of .6. The FACL6 protein was also expressed as a C-terminal histidine-tagged fusion protein in the pBAD/Myc-His vector (Daily life Systems, NY) below the control of the araBAD promoter in E. coli fadLfadR mutant LS6164 [twenty, 21]. All subcloning methods were carried out in pCR Blunt II TOPO vector (Existence Systems, NY) in E. coli TOP10 cells (Lifestyle Technologies, NY). Expression of FACL6 in E. coli mutant LS6164 at OD600 of .6 was induced for 4 h by the addition of arabinose to a ultimate focus of .two% (w/v).
Alignment of FACL6 amino acid sequence with eukaryotic FATPs and purification of FACL6. A, The FACL6 (Rv1206) amino BIX02189 structureacid sequence was aligned with human FATP1 (HsFATP1), human FATP4 (HsFATP4) and yeast FATP (ScFAT) amino acid sequences making use of the ClustalW2 many sequence alignment system. Amino acid identities are shaded in black and similarities shaded in grey. The diagonally hatched containers previously mentioned the amino acid sequences show the place of the AMP-binding domain and the vertically hatched containers reveal the site of the really prolonged-chain acyl-CoA synthetase (VLACS) area on the human FATP1. The peptide indicated by a box and arrow at the C-terminal conclusion of Rv1206 (FACL6) was used to increase a polyclonal antiserum towards FACL6. B, Purification of FACL6 expressed in E. coli. The FACL6 protein was solubilized and affinity-purified as explained in Elements and Procedures. Coomassie-stained denaturing polyacrylamide gel proven. Lanes: 1 and five, molecular body weight specifications (dimensions indicated on left) 2, cell lysate three, sixteen,0006g supernatant four, supernatant from solubilized sixteen,000 x g pellet six, column movement-through 7 and eight, column wash 9 and 10, eluted fractions that contains purified FACL6 protein.
Expression of FACL6 in pET200 D-TOPO build was induced with IPTG in a 600 ml tradition of E. coli BL21 Star (DE3). The cells were being washed and resuspended in lysis buffer (50 mM sodium phosphate pH 7.four, three hundred mM NaCl with a cocktail of protease inhibitors) and have been disrupted by sonication working with a Branson Sonifier 450 (Branson Ultrasonics Corp.). The mobile lysates have been clarified by centrifugation at sixteen,000 x g, four and the pellet was utilised for purification of the expressed protein considering that our makes an attempt to purify the protein from supernatant were unsuccessful. The pellet was solubilized in five ml ice-cold lysis buffer with 1% (w/v) dodecylmaltoside and .03% Triton X-one hundred for 15 min with intermittent sonication to boost solubilization of the inclusion bodies. The answer was centrifuged and the supernatant was utilized to purify the FACL6 protein working with a five-ml mattress volume TALON cobalt-affinity resin (Clontech, CA) in accordance to the manufacturer’s protocol. The column was washed with ten mM imidazole and bound protein was eluted with two hundred mM imidazole.Sennoside The proteins in each fraction were settled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by coomassie staining.
The purified FACL6 was assayed for acyl coenzyme-A (acyl-CoA) synthetase action pursuing a modified protocol of Nambi et al. [22]. Briefly, the assay mixture, in a 40 ml reaction quantity, contained about 7.five mg purified FACL6 protein, 100 mM 14C-oleic acid (or 14C-palmitic acid or 14C-stearic acid or 14Chexacosanoic acid), five mM ATP, 2 mM CoA, 2.5 mM MgCl2, .005% (v/v) Triton X-100 in fifty mM Tris-HCl pH eight.. Immediately after incubation for fifteen min at thirty, the response was stopped by addition of two.five ml glacial acetic acid. Acyl-CoA item migrated with an Rf of .55 although fatty acid substrate migrated with an Rf of one. (in the vicinity of the solvent entrance). The TLC plate was imaged by autoradiography and radioactivity in acyl-CoA band was calculated by liquid scintillation counting to acquire a quantitative evaluate of acyl-CoA synthetase action. Acyl-CoA synthetase activity in mobile-cost-free extracts (prepared by bead-beating Mtb cells from ten ml cultures in .five ml ice-cold 100 mM Tris-HCl pH seven.five made up of protease inhibitor cocktail) of Mtb subjected to dormancy-inducing several strain was assayed utilizing a protocol modified from Hall and colleagues [23].
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