Plasmids encoding the His-Alkbh4, His-HARE-HTH and His-RAMA domains were transformed into E. coli strain Rosetta 2 (DE3) (MilliporeSigma). Bacterial cultures were grown to an A600 of 0.4-0.5, then induced with 0.1 mM IPTG for 4 h at 30°C. Bacterial pellets were resuspended in ice-cold PBS supplemented with 100mM NaCl, 1mM Imidazole, 0.2% NP-40, complete protease inhibitor cocktail, 5 mM sodium fluoride, 1 mM sodium orthovanadate, lysozyme (0.2 mg/mL) and DNase I (10 μg/mL), then sonicated in 5-6 bursts of 15 s with 1 min cooling in between bursts. Soluble proteins (5 ml) were incubated with 250 μL Profinity Nickel-charged resin (Bio-Rad) for 1 h at 4°C with gentle rotation. Resin was washed twice in lysis buffer and eluted for 30 min in elution buffer (1XPBS, 100mM NaCl, 250mM Imidazole, 5% glycerol), then pooled and dialyzed overnight at 4°C into 1XTBS containing 5% glycerol. Samples were aliquoted and stored at −80°C.
Rosetta 2 de3
Manufactured by Merck Group
Sourced in Germany
Rosetta 2(DE3) is a bacterial expression system designed for the production of recombinant proteins in Escherichia coli. It provides tRNAs for rare codons found in eukaryotic genes, enabling the efficient expression of proteins that may be difficult to express in standard E. coli strains.
Automatically generated - may contain errors
Lab products found in correlation
Hiload 16 60 superdex 75 column, by GE Healthcare (1 mentions)
Akta pure, by Cytiva (1 mentions)
Np 40, by Merck Group (1 mentions)
E coli strain jm109, by Promega (1 mentions)
E coli bl21 de3, by Agilent Technologies (1 mentions)
Prare2 cam r, by Merck Group (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Pet28b, by Merck Group (1 mentions)
Bl21 codonplus de3 ripl chemically competent cells, by Agilent Technologies (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Nickel nitrilotriacetic acid agarose, by Qiagen (1 mentions)
Dodecyl β d maltopyranoside ddm, by Thermo Fisher Scientific (1 mentions)
Pet23a, by Merck Group (1 mentions)
Pet28a vector, by Merck Group (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Hitrap nhs activated hp column, by GE Healthcare (1 mentions)
Amicon ultra 4, by Merck Group (1 mentions)
Hitrap, by Cytiva (1 mentions)
Pet 49b, by Merck Group (1 mentions)
Pgex 6p 3, by GE Healthcare (1 mentions)
15 protocols using rosetta 2 de3
1
Purification of Recombinant Histone Demethylase Proteins
2
Purification and Analysis of Laforin Variants
3
Purification of Recombinant His-tagged MS2 Protein
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The expression plasmids encoding the His-tag fused MS2 protein gene were transformed into Rosetta 2(DE3) (Merck, Darmstadt, Germany). LB culture (100 mL) supplemented with 200 µg/mL ampicillin and 35 µg/mL chloramphenicol was inoculated with 1/100 volume of pre-cultured transformed cells and cultured at 37 °C until OD600 = 0.5. The recombinant proteins were induced with 0.1 mM IPTG at a final concentration of 15 °C for 16 h with shaking. After harvesting and centrifugation, the cell pellets were dissolved in ice-cold extraction buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40 (Sigma-Aldrich, St. Louis, MO, USA), 1 mM PMSF, 2 mg/mL lysozyme, and 2 µL 10 mg/mL DNase) and then sonicated. Cell debris was removed after centrifugation (twice at 15,000× g, 10 min, 4 °C). Half of the lysate solution was stored at −80 °C until use. For purification of His-tagged MS2 protein, 200 µL Ni-NTA slurry beads (QIAGEN, Venlo, The Netherlands) were added to the lysate, incubated with rotation at 4 °C for 1 h, and washed three times with wash buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.5% NP-40, and 10 mM imidazole). The recombinant protein was eluted using elution buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.5% NP-40, and 500 mM imidazole) and was dialyzed at 4 °C overnight in dialysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, 1 mM DTT, and 1 mM EDTA).
4
Cultivation of Bacterial Strains
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Bacillus clausii DSM8716, Bacillus coagulans DSM1, Marivirga tractuosa DSM4126, Spirosoma linguale DSM74 and Streptomyces pristinaespiralis DSM40338 were obtained from the German collection of microorganisms and cultivated according to the instructions of the supplier (DSMZ, http://www.dsmz.de ). E. coli strain JM109 [genotype endA1 recA1, gyrA96, thi, hsdR17,(rK−, mK+), relA1, supE44, l-, Δ(lac-proAB), (F’, traD36, proAB, lacIqZΔM15)] was purchased from Promega (Madison, USA). E. coli BL21(DE3) was purchased from Agilent Technologies. Rosetta™ 2(DE3) [genotype F– ompT hsdSB(rB– mB–) gal dcm (DE3) pRARE2 (CamR)] was purchased from Merck Millipore (Germany).
E. coli strains were routinely grown in LB medium at 37 °C and 150 rpm. For plasmid selection 100 mg L−1 ampicillin was added to LB agar plates and liquid medium and 34 μg/mL chloramphenicol when cells were co-transformed with pRARE2.
E. coli strains were routinely grown in LB medium at 37 °C and 150 rpm. For plasmid selection 100 mg L−1 ampicillin was added to LB agar plates and liquid medium and 34 μg/mL chloramphenicol when cells were co-transformed with pRARE2.
5
Generating Anti-PNG Antibodies
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His-tagged full length PNG protein was expressed in Rosetta 2(DE3) (EMD Millipore, Billerica, MA) using pET28b (EMD Millipore, Billerica, MA) and purified with Ni-NTA Agarose (Qiagen, Valencia, CA) under denaturing condition. Purified His-PNG was injected to rabbits to raise antibodies (Covance, Princeton, NJ). The specificity of the antibody was confirmed by western blots of png mutant extracts.
6
Recombinant TDP1 Catalytic Domain
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A codon-optimised synthetic gene, encoding the catalytic domain of human TDP1, was purchased from GenScript USA Inc. Details of the expression construct are consistent with that described in Interthal et al. [6] (link). Recombinant protein was expressed in, then purified from Escherichia coli strain Rosetta2 (DE3) (Merck, Darmstadt, Germany) using standard chromatography techniques, following the protocol described by Interthal et al. [6] (link).
7
Cloning and Expression of GsI-IIC RT
8
Heterologous Expression of Chryseobacterium gleum
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Chryseobacterium gleum DSM 16776 was obtained from the German collection of microorganisms (DSMZ). E. coli strain JM109 [genotype endA1 recA1, gyrA96, thi, hsdR17,(rK-, mK+), relA1, supE44, λ-, Δ(lac-proAB), (F', traD36, proAB, lacIqZΔM15)] and the pQE-30 expression vector were purchased from Promega (Madison, USA) and Qiagen (Valencia, USA), respectively. The origin of replication in pQE-30 is ColE1 (pBR322) and transcription of the inserted gene is controlled by the bacteriophage T5 promoter (recognized by the E. coli housekeeping RNA polymerase) and two lac operator sequences (conferring inducibility by IPTG). For efficient repression the host strain JM109 which over-expresses the LacI repressor was used. JM109 was transformed with the plasmid pRARE2, which contains the tRNA genes argU, argW, ileX, glyT, leuW, proL, metT, thrT, tyrU, thrU and argX. The usage of the rare codons AGG, AGA, AUA, CUA, CCC, GGA and CGG is thereby supplemented. The plasmid was isolated from Rosetta2 (DE3) (Merck Chemicals, UK) (F-ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE2) cells. The resulting chloramphenicol-resistant strain JM109-pRARE2 was the expression host.
9
Cloning and Purification of Borrelia OMP Proteins
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Candidate OMP DNA sequences including bb0405, bb0406, and bb0838 were amplified from B. burgdorferi B31 genomic DNA using primers listed in Table 3 . The amplicons were subsequently digested and cloned into the NheI or BamHI and XhoI sites of pET23a (EMD Millipore, Billerica, MA). The constructs were transformed into the E. coli strain Rosetta 2 DE3 (EMD Millipore), and DNA sequencing was performed to verify that the sequence remained unaltered throughout the cloning process. Recombinant proteins were induced and purified using nickel-nitrilotriacetic acid agarose (Qiagen,Valencia, CA) as described previously [51 (link)]. Recombinant BB0405 and BB0406 were folded in DDM buffer [50 mM Tris, 100 mM NaCl, dodecyl-β-D-maltopyranoside (DDM; Affymetrix, 14 Santa Clara, CA)] pH 7.6 for BB0405 (0.5 % DDM) and pH 8.6 for BB0406 (2.0 % DDM) (pH and DDM concentrations were optimized for each protein) at 4 °C overnight, and the insoluble material was pelleted by centrifugation at 20,000 x g for 30 min at 4 °C.
10
Autotrophic Growth of M. sedula TH2T
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Cells of M. sedula TH2T (DSM 5348) were grown autotrophically at 75°C modified Allen medium, pH 2.0 (Huber and Prangishvili, 2006 (link)) in a 100-l fermenter under gassing with a mixture of 19% CO2, 3% O2, and 78% H2 (0.5 l/min) (generation time, 8 h) (Huber et al., 1989 (link), 1992 (link)). Cells were frozen in liquid nitrogen and stored at −80°C until use. Escherichia coli strain DH5α and E. coli strain Rosetta 2 (DE3) (Merck, Germany) were grown at 37°C in lysogeny broth (LB) medium. Antibiotics were added to the cultures to a final concentration of 100 μg ampicillin ml–1 and 34 μg chloramphenicol ml–1.
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