Mle genomic DNA was obtained from the Thai-53 strain45 (link) maintained at the Leprosy Research Center, National Institute of Infectious Diseases (Tokyo, Japan). E. coli Top-10 (Life Technologies) and DH5α were used as hosts for molecular cloning, whereas strains Rosetta-gami2 (DE3) pLysS and BL21 (DE3) pLysS were used to express Mle and Mtb GyrA and GyrB subunits from plasmids pET-20b (+) and pET-19b (Merck KGaA, Darmstadt, Germany). Msmeg mc2 155 was used as the mycobacterial host to produce strains expressing DNA gyrase from Mle and Mavi 104 strains. The cosmid vector pYUB854 and plasmid vector phAE87, which were used to construct vectors for allelic exchange, were kindly provided by W. R. Jacobs, Jr. (Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY, USA).
Pet19b
Manufactured by Merck Group
Sourced in Germany
PET19b is a laboratory instrument designed for the purification and analysis of proteins. It utilizes affinity chromatography to capture and separate proteins of interest from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Pet 20b, by Merck Group (2 mentions)
Bl21 de3 plyss, by Merck Group (2 mentions)
E coli top10, by Thermo Fisher Scientific (1 mentions)
Pet22b, by Merck Group (1 mentions)
E coli jm109, by Takara Bio (1 mentions)
Birchwood xylan, by Merck Group (1 mentions)
Pgex4t1, by Merck Group (1 mentions)
Ni nta, by Qiagen (1 mentions)
Cnbr activated sepharose, by GE Healthcare (1 mentions)
Bl21 codonplus de3 ril, by Agilent Technologies (1 mentions)
Pet26b, by Merck Group (1 mentions)
Pet26b, by Agilent Technologies (1 mentions)
Bl21 de3 plyss, by Promega (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Pmal c5x, by New England Biolabs (1 mentions)
7 protocols using pet19b
1
Molecular Cloning of Mycobacterial Gyrase
2
Xylanase Production by Paenibacillus curdlanolyticus
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Paenibacillus curdlanolyticus B-6 is in the Culture Collection of the National Center for Genetic Engineering and Biotechnology, Thailand (accession number BCC 11175). Strain B-6 was grown on Bergey’s mineral salt medium (pH 7.0) supplemented with birchwood xylan (Sigma-Aldrich, St. Louis, MO, USA) as previously reported (Pason et al. 2006 (link), 2010 (link)). E. coli JM109 (TaKaRa Bio, Shiga, Japan), E. coli Rosetta™ 2 (DE3), E. coli BL21 (DE3) (Merck KGaA, Darmstadt, Germany), pET19b, and pET22b (Merck KGaA) served as cloning and expression hosts and protein expression vectors.
3
Expressing GST-tagged Ly49Q Mutants in E. coli
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To express the Glutathione S-Transferase (GST)-tagged Ly49Q mutants in E. coli, DNA corresponding to the mutant proteins indicated was amplified by PCR and cloned and inserted into pGEX4T1 (GE Healthcare Technologies Inc.). To express GST- or His6-tagged fusion proteins in E. coli, DNA corresponding to the SH2 domains of the phospho-ITIM-interacting proteins was amplified by PCR and cloned and inserted into pGEX4T1 or pET19b (Merck Millipore Co.). The expressed SH2 domains were as follows: mouse CrkL (residues 1–110), mouse Lyn (residues 101–250), mouse Shc (residues 451–579), mouse Grb2 (residues 41–190), mouse Csk (residues 50–200), mouse Hck (residues 100–250), mouse Fgr (residues 100–250), and mouse PLCγ2 (residues 501–640, 501–750, and 625–750). Mouse Grb2 and Ly49Q were cloned and inserted into pCAG-HA or pCAGGS-EGFP expression vectors.
4
Recombinant Zscan4 Antibody Production
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Polyclonal antibodies against mouse Zscan4 (a.a.1-506) were produced by inserting its complementary DNA (cDNA) fragment in-frame with pET19b (Merck Millipore , Tokyo Japan) in E. coli strain BL21-CodonPlus(DE3). His-tagged recombinant proteins were solubilized in a denaturing buffer (6 M HCl-Guanidine, 20 mM Tris-HCl [pH 7.5]) from the inclusion body and purified by Ni-NTA (QIAGEN, Tokyo Japan) under denaturing conditions. After dialyzing against PBS, the purified protein was used to immunize rats and rabbits. The antibodies were affinity purified from the immunized crude serum with immobilized antigen on CNBr-activated Sepharose (GE Healthcare, Tokyo Japan).
5
Cloning and Purification of PqsB and PqsC
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The pqsB (PA0997) and pqsC (PA0998) genes from Pseudomonas aeruginosa PAO1 were amplified from chromosomal DNA by PCR (primers listed in Table S4). pqsB was cloned into pET26b (Merck Millipore) and pqsC was ligated into pET19m or p10$, [71] both based on the pET19b vector (Merck Millipore). The resulting plasmid pET19m-pqsC produces PqsC with an Nterminal His6 tag followed by a recognition motif for TEV (tobacco etch virus) protease, while p10$-pqsC encodes PqsC with an N-terminal His6-tagged T7 lysozyme removable by human rhinovirus 3C protease. The active site cysteine C129 of PqsC was mutated to alanine (PqsC C129A ) or serine (PqsC C129S ) in pET19mod-pqsC and p10$-pqsC by PCR-based mutagenesis (Table S4).
Expression and purification of (His6-)PqsBC, (His6-)PqsBC C129A and PqsBC C129S Recombinant proteins were produced in E. coli BL21(DE3)pLysS (Promega) or BL21-CodonPlus(DE3)-RIL (Agilent Technologies) co-transformed with pET26b-pqsB and pET19m-pqsC or p10$-pqsC. Purification involved nickel affinity and size exclusion chromatography with or without an intermittent protease cleavage and chromatography step to remove the His6-affinity tag (Table S5). The purified proteins were concentrated to 20 -35 mg mL -1 , flash-cooled in liquid nitrogen and stored at -80 °C.
Expression and purification of (His6-)PqsBC, (His6-)PqsBC C129A and PqsBC C129S Recombinant proteins were produced in E. coli BL21(DE3)pLysS (Promega) or BL21-CodonPlus(DE3)-RIL (Agilent Technologies) co-transformed with pET26b-pqsB and pET19m-pqsC or p10$-pqsC. Purification involved nickel affinity and size exclusion chromatography with or without an intermittent protease cleavage and chromatography step to remove the His6-affinity tag (Table S5). The purified proteins were concentrated to 20 -35 mg mL -1 , flash-cooled in liquid nitrogen and stored at -80 °C.
6
Production and Characterization of TAT-Fused Proteins
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Plasmids used were previously described [10 (link), 11 (link)] or constructed as described in (S1 Fig ). Briefly, pJM161 consisted of an E. coli-optimized synthetic gene (GeneScript, Piscataway, NJ, USA) encoding TAT-naked mole rat calmodulin (TAT-NMR-CaM) cloned into NdeI and BamHI sites in pET19b (EMD Millipore, USA) with an in-frame stop codon prior to the BamHI site. The encoded TAT-NMR-CaM protein consists of the TAT peptide sequence (YGRKKRRQRRR) N-terminally fused to Heterocephalus glaber (naked mole rat) calmodulin (GenBank: EHB02604.1) [19 (link)]. A vector-encoded 10xHis tag is N-terminal to TAT. Plasmids pJM140 and pJM168, encoding CBS-maltose binding protein (CBS-MBP) and TAT-maltose binding protein (TAT-MBP), respectively, were made by cloning synthetic gene fragments encoding the CBS or TAT peptide sequences into NdeI and BamHI sites in pMAL-c5x (New England Biolabs, Ipswich, MA). The encoded cargo proteins thus have either a CBS or TAT sequence C-terminal to the MBP and a 6x His tag beyond.
E. coli used in this study, BL21(DE3)pLysS was propagated from purchased cells from EMD Millipore (Burlington, MA, USA) or other established supplier.
Baby hamster kidney (BHK) cells were purchased from ATCC (#CCL-10) and cultured in Dulbecco’s Modified Eagles’ Medium with GlutaMAX Supplement (Gibco, USA) and 10% fetal bovine serum.
E. coli used in this study, BL21(DE3)pLysS was propagated from purchased cells from EMD Millipore (Burlington, MA, USA) or other established supplier.
Baby hamster kidney (BHK) cells were purchased from ATCC (#CCL-10) and cultured in Dulbecco’s Modified Eagles’ Medium with GlutaMAX Supplement (Gibco, USA) and 10% fetal bovine serum.
7
Mycobacterium tuberculosis Genetic Manipulation
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Mtb var. bovis BCG Tokyo 172 strains were provided from the Japan BCG Laboratory (Tokyo, Japan). FQ-susceptible or -resistant Mtb Beijing lineage strains listed in Supplementary Table S2 were obtained from Osaka Institute of Public Health (Osaka, Japan). E. coli strain DH5α (Takara Bio Inc.) was used for DNA cloning. E. coli strains Rosetta-gami 2 (DE3) and BL21 (DE3) pLysS (Merck KGaA, Darmstadt, Germany) were used for protein expression. Vector plasmids pET-20b (+) and pET-19b (Merck KGaA) were used to construct plasmids for the expression of Mtb GyrA and GyrB, respectively. A Mtb GyrB expression plasmid, pTB-B, was constructed in our previous study [10] (link). A parental plasmid for allelic exchange, pΔAHm31, was constructed, as shown in Supplementary Fig. S1. Recombinase expression plasmid, pJV53, and resolvase expression plasmid, pYUB870 were gifts from Dr. Hatfull and Dr. Jacobs, respectively [11, (link)12] .
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