escherichia coli strain bl21  (Millipore)


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    Structured Review

    Millipore escherichia coli strain bl21
    Escherichia Coli Strain Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/escherichia coli strain bl21/product/Millipore
    Average 99 stars, based on 181 article reviews
    Price from $9.99 to $1999.99
    escherichia coli strain bl21 - by Bioz Stars, 2020-01
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Disabling Cas9 by an anti-CRISPR DNA mimic
    Article Snippet: Phage-encoded anti-CRISPR AcrIIA4 ( ) was subcloned using Clontech’s In-Fusion cloning system into a pGEX-6P-1 expression vector with a glutathione S -transferase (GST) affinity tag and a PreScission Protease cleavage site at the N terminus. .. The GST-AcrIIA4 fusion protein was overexpressed in Escherichia coli strain BL21 (Novagen) with overnight induction of 0.25 mM isopropyl-β-d -thiogalactopyranoside at 18°C.

    Article Title: Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6
    Article Snippet: Paragraph title: Cloning of PSF protein, truncated RBD and DBD protein, expression and purification of recombinant proteins ... The plasmid was transformed into Escherichia coli strain BL21 (Novagen).

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: .. Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen). ..

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The Hin dIII-EGFP- Xho I and Nco I-TAT- Hin dIII PCR fragments were cloned into pET28a(+) to prepare the pET28a-TAT-hMT1A and pET28a-CAMP-hMT1A plasmids, respectively. .. The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA).

    Article Title: Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery
    Article Snippet: The full-length AtEH1/Pan1 cDNA was cloned into the SacI/XhoI sites of the pET42a vector (with N-terminal GST and HisTag) for protein expression. .. The construct was transformed into E. coli strain BL21 (Novagen), and protein expression was induced by IPTG (1 mM) at 160 overnight.

    Centrifugation:

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark). .. After centrifugation (19,000 × g, 20 min, at 4°CC), the supernatants were applied to a 5 mL nickel column (Bio-Scale Mini Profinity IMAC Cartridges) for Ii049 recombinant protein purification according to the manufacturer's recommendation (Cat. #732-4612, Bio-Rad Laboratories, USA).

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking. .. Protein expression was induced by the addition of isopropyl β- d -1-thiogalactopyranoside (IPTG) to a final concentration of 1 m M , followed by incubation with shaking for an additional 3 h. Cells were harvested by centrifugation, frozen using liquid nitrogen and stored at 193 K until needed.

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen). .. Cells were harvested after 16 h, and resuspended in phosphate-buffered saline (PBS) solution (10 mM Na2 HPO4 , 1.8 mM KH2 PO4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4) supplemented with 350 mM NaCl, disrupted by sonication, and the resulting cell lysates were clarified by centrifugation and filtration.

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA). .. Cell pellets were collected by centrifugation and suspended in lysis buffer (300 mM KCl, 49 mM KH2 PO4 , and 4.9 mM imidazole) in the presence of 100 mM phenylmethylsulfonyl fluoride (PMSF), followed by sonication for eight cycles of 30 s each .

    Article Title: Mechanism of microtubule stabilization by taccalonolide AJ
    Article Snippet: Protein expression and purification The stathmin-like domain of RB3 (RB3-SLD) and TTL were overexpressed in Escherichia coli strain BL21(DE3) (Novagen). .. Cells expressing RB3-SLD were collected by centrifugation and resuspended in loading buffer (20 mM Tris-HCl (pH 8.0), 1 mM EGTA, 2 mM dithiothreitol (DTT)) and then lysed by an ultrahigh-pressure homogenizer (JNBIO).

    Amplification:

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: Electrophoretic mobility shift assay The full length coding sequence of Ii049 was amplified with the primers Ii049 -pET-F and Ii049 -pET-R (Supplementary Table ), and inserted into the expression vector pET32a (Novagen, Denmark) between the Nco I and Hind III sites for the generation of Ii049 recombinant protein. .. The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark).

    Article Title: Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6
    Article Snippet: Cloning of PSF protein, truncated RBD and DBD protein, expression and purification of recombinant proteins The PSF open reading frame was PCR amplified from HEK293 cDNA with primers: P5: 5’- CTAGTATGTCTCGGGATCGGTTCC-3’ and P6: 5’-CAGCTACTCGA CTAAAATCGGGGTTTTTTGTTTG-3’ ; Primers for truncated DBD (1–97 aa): P9 5’- CTAGTATGTCTCGGGATCGGTTCC-3’ and P10 : 5’-CAGCTACTCGATGCTGCTGATGCGGCTGTGGATG-3’ ; Primers for truncated RBD (98–366 aa): P7 5’- CTAGTATGGCAGCCGCCGCCACCGCCG-3’ and P8: 5’-CAGCTACTCGACAAAGCGAACTCGAAGCTGTCT-3’ . .. The plasmid was transformed into Escherichia coli strain BL21 (Novagen).

    Filtration:

    Article Title: Disabling Cas9 by an anti-CRISPR DNA mimic
    Article Snippet: The GST-AcrIIA4 fusion protein was overexpressed in Escherichia coli strain BL21 (Novagen) with overnight induction of 0.25 mM isopropyl-β-d -thiogalactopyranoside at 18°C. .. The recovered proteins were then digested with PreScission Protease to remove the GST tag and further fractionated by ion exchange column (Hitrap Q), followed by an additional gel filtration chromatography step (HiLoad 16/60 Superdex 200, GE Healthcare) using the storage buffer [50 mM Hepes (pH 7.5), 200 mM NaCl, 5 mM dithiothreitol (DTT), and 10% glycerol].

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen). .. Cells were harvested after 16 h, and resuspended in phosphate-buffered saline (PBS) solution (10 mM Na2 HPO4 , 1.8 mM KH2 PO4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4) supplemented with 350 mM NaCl, disrupted by sonication, and the resulting cell lysates were clarified by centrifugation and filtration.

    Synthesized:

    Article Title: Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR
    Article Snippet: Viral polymerases The DNA sequences of previously identified viral metagenomic polymerases ( ) were obtained from GenBank ( AFN99405.1 , AGL03982 , AGL03983 , AGL03985 AGL03987 and AGL03990 ), altered to eliminate 3′→5′ exonuclease activity as previously described , codon optimized, and synthesized by Synthetic Genomics. .. The DNA constructs were then assembled with a tac promoter-based expression vector and used to transform Escherichia coli strain BL21 (EMD Millipore).

    Construct:

    Article Title: Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR
    Article Snippet: .. The DNA constructs were then assembled with a tac promoter-based expression vector and used to transform Escherichia coli strain BL21 (EMD Millipore). .. Magma fusion The 5′→3′ nuclease domain of the Taq polymerase gene (GenBank BAA06775 ) was PCR amplified using Veraseq 2.0 polymerase (QIAGEN) and primers Taqexo-F and Taqexo-R.

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: These constructs produce recombinant proteins bearing an N-terminal hexahistidine tag that can be removed by cleavage with thrombin, leaving the residual sequence GSH– at the N-termini of the mature proteins. .. Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking.

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: .. Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen). ..

    Article Title: Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery
    Article Snippet: .. The construct was transformed into E. coli strain BL21 (Novagen), and protein expression was induced by IPTG (1 mM) at 160 overnight. .. GST-His-Tagged fusion protein was purified using Ni-NTA resin (Qiagen) according to manufacturer’s instructions.

    Article Title: Curcumin protects mice from Staphylococcus aureus pneumonia by interfering with the self-assembly process of α-hemolysin
    Article Snippet: Mutagenesis of the Hla protein The recombinant plasmids encoding WT-Hla and its two mutants, Q89A-Hla and K163A-Hla, were constructed as described in our previous work . .. After transforming the recombinant plasmids into Escherichia coli strain BL21(DE3) (Novagen), the soluble Hla proteins were obtained from induced E. coli cells under the appropriate conditions for high-level expression and purification of recombinant Hla protein described by Qiu et al .

    Incubation:

    Article Title: Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy
    Article Snippet: Recombinant proteins purification T7‐His‐tagged ATG16L172–307 and mutants were expressed from pET28a plasmid in Escherichia coli strain BL21 (DE3, Novagen) and grown overnight at 37°C in 50 ml of lysogeny broth (LB) media, containing 50 μg/ml kanamycin. .. After the initial incubation, the cell culture was diluted into 800 ml LB media with the selection agent and incubated at 37°C until OD600 reached 0.8.

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking. .. Protein expression was induced by the addition of isopropyl β- d -1-thiogalactopyranoside (IPTG) to a final concentration of 1 m M , followed by incubation with shaking for an additional 3 h. Cells were harvested by centrifugation, frozen using liquid nitrogen and stored at 193 K until needed.

    Article Title: Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery
    Article Snippet: The construct was transformed into E. coli strain BL21 (Novagen), and protein expression was induced by IPTG (1 mM) at 160 overnight. .. Rabbit muscle actin (Cytoskeleton Inc.) suspended in G-buffer (2 mM Tris pH 8.0, 0.5 mM DTT, 0.2 mM CaCl2 , 0.2 mM ATP) was polymerized by the addition of 10× KME (500 mM KCl, 10 mM MgSO4 , 10 mM EGTA, 100 mM imidazole, pH 6.5) and incubated at room temperature for 2 h. Meanwhile, AtEH1/Pan1 protein solution was centrifuged at 500,000 × g for 30 min to remove any insoluble fractions that may interfere with the experiments.

    Activity Assay:

    Article Title: Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR
    Article Snippet: Viral polymerases The DNA sequences of previously identified viral metagenomic polymerases ( ) were obtained from GenBank ( AFN99405.1 , AGL03982 , AGL03983 , AGL03985 AGL03987 and AGL03990 ), altered to eliminate 3′→5′ exonuclease activity as previously described , codon optimized, and synthesized by Synthetic Genomics. .. The DNA constructs were then assembled with a tac promoter-based expression vector and used to transform Escherichia coli strain BL21 (EMD Millipore).

    Expressing:

    Article Title: Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR
    Article Snippet: .. The DNA constructs were then assembled with a tac promoter-based expression vector and used to transform Escherichia coli strain BL21 (EMD Millipore). .. Magma fusion The 5′→3′ nuclease domain of the Taq polymerase gene (GenBank BAA06775 ) was PCR amplified using Veraseq 2.0 polymerase (QIAGEN) and primers Taqexo-F and Taqexo-R.

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: Electrophoretic mobility shift assay The full length coding sequence of Ii049 was amplified with the primers Ii049 -pET-F and Ii049 -pET-R (Supplementary Table ), and inserted into the expression vector pET32a (Novagen, Denmark) between the Nco I and Hind III sites for the generation of Ii049 recombinant protein. .. The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark).

    Article Title: Disabling Cas9 by an anti-CRISPR DNA mimic
    Article Snippet: Paragraph title: Protein expression and purification ... The GST-AcrIIA4 fusion protein was overexpressed in Escherichia coli strain BL21 (Novagen) with overnight induction of 0.25 mM isopropyl-β-d -thiogalactopyranoside at 18°C.

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: Paragraph title: 2.1. Protein expression and purification   ... Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking.

    Article Title: Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6
    Article Snippet: Paragraph title: Cloning of PSF protein, truncated RBD and DBD protein, expression and purification of recombinant proteins ... The plasmid was transformed into Escherichia coli strain BL21 (Novagen).

    Article Title: Genetically designing a more potent anti-pancreatic cancer agent by simultaneously cotargeting human IL-13 and EGF receptors in a mouse xenograft model
    Article Snippet: Plasmids were transformed into Escherichia coli strain BL21(DE3) (Novagen, Madison WI). .. Gene expression was induced with the addition of IPTG (FischerBiotech, Fair Lawn, NJ).

    Article Title: Heterodimeric bispecific single chain variable fragments (scFv) killer engagers (BiKEs) enhance NK-cell activity against CD133+ colorectal cancer cells
    Article Snippet: .. For bacterial protein expression, plasmids were transformed into Escherichia coli strain BL21(DE3) (Novagen, Madison WI). .. Following overnight culture, bacteria were grown in 800 ml Luria broth containing 50 mg/ml carbenicillin.

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: Paragraph title: Protein recombinant expression and purification ... Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen).

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The synthetic Nco I-CAMP- Hin dIII-hMT1A- Xho I gene (Bioneer) was cloned into the pET28a(+) vector (Clontech) to prepare the pET28a-CAMP-MT1A bacterial expression plasmid. .. The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA).

    Article Title: Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery
    Article Snippet: .. The construct was transformed into E. coli strain BL21 (Novagen), and protein expression was induced by IPTG (1 mM) at 160 overnight. .. GST-His-Tagged fusion protein was purified using Ni-NTA resin (Qiagen) according to manufacturer’s instructions.

    Article Title: Curcumin protects mice from Staphylococcus aureus pneumonia by interfering with the self-assembly process of α-hemolysin
    Article Snippet: .. After transforming the recombinant plasmids into Escherichia coli strain BL21(DE3) (Novagen), the soluble Hla proteins were obtained from induced E. coli cells under the appropriate conditions for high-level expression and purification of recombinant Hla protein described by Qiu et al . .. Fluorescence-quenching assay In our paper, fluorescence quenching can be described by the following Scatchard equation: r /D f = nK − rK , where r is the ligand amount of substance per mole of protein binding (r ≈ ΔF /F 0 ), D f is the free concentration of CUR, K is the binding constant and n is the number of binding sites.

    Article Title: Mechanism of microtubule stabilization by taccalonolide AJ
    Article Snippet: .. Protein expression and purification The stathmin-like domain of RB3 (RB3-SLD) and TTL were overexpressed in Escherichia coli strain BL21(DE3) (Novagen). .. Cells expressing RB3-SLD were collected by centrifugation and resuspended in loading buffer (20 mM Tris-HCl (pH 8.0), 1 mM EGTA, 2 mM dithiothreitol (DTT)) and then lysed by an ultrahigh-pressure homogenizer (JNBIO).

    Western Blot:

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark). .. Protein integrity was checked by western blotting using primary anti-His-Tag antibody (Cat. #12698, Cell Signaling Technology, USA) and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Cat. #7074, Cell Signaling Technology, USA).

    Transformation Assay:

    Article Title: Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6
    Article Snippet: .. The plasmid was transformed into Escherichia coli strain BL21 (Novagen). .. His-tagged PSF protein was expressed in 200 ml Overnight Express medium (Novagen) for 24h at 25°C.

    Article Title: Genetically designing a more potent anti-pancreatic cancer agent by simultaneously cotargeting human IL-13 and EGF receptors in a mouse xenograft model
    Article Snippet: .. Plasmids were transformed into Escherichia coli strain BL21(DE3) (Novagen, Madison WI). ..

    Article Title: Heterodimeric bispecific single chain variable fragments (scFv) killer engagers (BiKEs) enhance NK-cell activity against CD133+ colorectal cancer cells
    Article Snippet: .. For bacterial protein expression, plasmids were transformed into Escherichia coli strain BL21(DE3) (Novagen, Madison WI). .. Following overnight culture, bacteria were grown in 800 ml Luria broth containing 50 mg/ml carbenicillin.

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: .. Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen). ..

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: .. The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA). .. The cells were then cultured in LB medium containing 50 μg/ml ampicillin at 37 °C for 4 h, and CAMP-hMT1A protein expression was induced by adding 1 mM isopropyl-β- d -thio-galactoside and incubating overnight at 26 °C.

    Article Title: Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery
    Article Snippet: .. The construct was transformed into E. coli strain BL21 (Novagen), and protein expression was induced by IPTG (1 mM) at 160 overnight. .. GST-His-Tagged fusion protein was purified using Ni-NTA resin (Qiagen) according to manufacturer’s instructions.

    Countercurrent Chromatography:

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The sequence of the synthetic gene was CC ATG G GC TAT GGC AGG AAG AAG CGG AGA CAG CGA CGA CGA TTG TTG CGC GCT GCC CTG CGC AAG GCT GCC CTG GGA AAG CTT ATG GAC CCC AAC TGC TCC TGC GCC ACT GGT GGC TCC TGC ACC TGC ACT GGC TCC TGC AAA TGC AAA GAG TGC AAA TGC ACC TCC TGC AAG AAG AGC TGC TGC TCC TGC TGC CCC ATG AGC TGT GCC AAG TGT GCC CAG GGC TGC ATC TGC AAA GGG GCA TCA GAG AAG TGC AGC TGC TGT GCC CTC GAG . .. The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA).

    Chromatography:

    Article Title: Disabling Cas9 by an anti-CRISPR DNA mimic
    Article Snippet: The GST-AcrIIA4 fusion protein was overexpressed in Escherichia coli strain BL21 (Novagen) with overnight induction of 0.25 mM isopropyl-β-d -thiogalactopyranoside at 18°C. .. The recovered proteins were then digested with PreScission Protease to remove the GST tag and further fractionated by ion exchange column (Hitrap Q), followed by an additional gel filtration chromatography step (HiLoad 16/60 Superdex 200, GE Healthcare) using the storage buffer [50 mM Hepes (pH 7.5), 200 mM NaCl, 5 mM dithiothreitol (DTT), and 10% glycerol].

    Concentration Assay:

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking. .. Protein expression was induced by the addition of isopropyl β- d -1-thiogalactopyranoside (IPTG) to a final concentration of 1 m M , followed by incubation with shaking for an additional 3 h. Cells were harvested by centrifugation, frozen using liquid nitrogen and stored at 193 K until needed.

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen). .. At OD600 of 0.6, growth temperature was shifted to 18°C and protein expression was induced by isopropyl-β-d -1-thiogalactopyranoside (IPTG; 0.5 mM final concentration).

    Protease Inhibitor:

    Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells
    Article Snippet: Recombinant protein purification The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA). .. Thereafter, the cells were harvested and washed twice with cold PBS, resuspended in PBS containing a protease inhibitor cocktail and lysed by sonication.

    Cell Culture:

    Article Title: Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy
    Article Snippet: Recombinant proteins purification T7‐His‐tagged ATG16L172–307 and mutants were expressed from pET28a plasmid in Escherichia coli strain BL21 (DE3, Novagen) and grown overnight at 37°C in 50 ml of lysogeny broth (LB) media, containing 50 μg/ml kanamycin. .. After the initial incubation, the cell culture was diluted into 800 ml LB media with the selection agent and incubated at 37°C until OD600 reached 0.8.

    Article Title: Heterodimeric bispecific single chain variable fragments (scFv) killer engagers (BiKEs) enhance NK-cell activity against CD133+ colorectal cancer cells
    Article Snippet: For bacterial protein expression, plasmids were transformed into Escherichia coli strain BL21(DE3) (Novagen, Madison WI). .. Two hours after induction, bacteria were harvested (from 4 liters cultured media we isolated 25g bacterial pellet) and then homogenized in a buffer solution (50 mM tris, 50 mM NaCl, and 5 mM EDTA pH 8.0).

    Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells
    Article Snippet: Recombinant protein purification The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA). .. Cells were cultured in Luria-Bertani medium and selected with ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml).

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA). .. The cells were then cultured in LB medium containing 50 μg/ml ampicillin at 37 °C for 4 h, and CAMP-hMT1A protein expression was induced by adding 1 mM isopropyl-β- d -thio-galactoside and incubating overnight at 26 °C.

    Sequencing:

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: Electrophoretic mobility shift assay The full length coding sequence of Ii049 was amplified with the primers Ii049 -pET-F and Ii049 -pET-R (Supplementary Table ), and inserted into the expression vector pET32a (Novagen, Denmark) between the Nco I and Hind III sites for the generation of Ii049 recombinant protein. .. The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark).

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: These constructs produce recombinant proteins bearing an N-terminal hexahistidine tag that can be removed by cleavage with thrombin, leaving the residual sequence GSH– at the N-termini of the mature proteins. .. Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking.

    Article Title: Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6
    Article Snippet: After confirmation by sequencing, the PSF full-length open reading frame was excised from plasmids with Nde I and Xho I, then cloned into similarly digested pET30b (Novagen) to produce pET30b-PSF, RBD or DBD separately. .. The plasmid was transformed into Escherichia coli strain BL21 (Novagen).

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The sequence of the synthetic gene was CC ATG G GC TAT GGC AGG AAG AAG CGG AGA CAG CGA CGA CGA TTG TTG CGC GCT GCC CTG CGC AAG GCT GCC CTG GGA AAG CTT ATG GAC CCC AAC TGC TCC TGC GCC ACT GGT GGC TCC TGC ACC TGC ACT GGC TCC TGC AAA TGC AAA GAG TGC AAA TGC ACC TCC TGC AAG AAG AGC TGC TGC TCC TGC TGC CCC ATG AGC TGT GCC AAG TGT GCC CAG GGC TGC ATC TGC AAA GGG GCA TCA GAG AAG TGC AGC TGC TGT GCC CTC GAG . .. The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA).

    Sonication:

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking. .. Cell lysis was initiated by adding hen egg-white lysozyme (Thermo Fisher) to a final concentration of 1 mg ml−1 and incubating on ice for ∼45 min, after which lysis was completed by sonication.

    Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells
    Article Snippet: Recombinant protein purification The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA). .. Thereafter, the cells were harvested and washed twice with cold PBS, resuspended in PBS containing a protease inhibitor cocktail and lysed by sonication.

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen). .. Cells were harvested after 16 h, and resuspended in phosphate-buffered saline (PBS) solution (10 mM Na2 HPO4 , 1.8 mM KH2 PO4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4) supplemented with 350 mM NaCl, disrupted by sonication, and the resulting cell lysates were clarified by centrifugation and filtration.

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA). .. Cell pellets were collected by centrifugation and suspended in lysis buffer (300 mM KCl, 49 mM KH2 PO4 , and 4.9 mM imidazole) in the presence of 100 mM phenylmethylsulfonyl fluoride (PMSF), followed by sonication for eight cycles of 30 s each .

    Recombinant:

    Article Title: Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy
    Article Snippet: .. Recombinant proteins purification T7‐His‐tagged ATG16L172–307 and mutants were expressed from pET28a plasmid in Escherichia coli strain BL21 (DE3, Novagen) and grown overnight at 37°C in 50 ml of lysogeny broth (LB) media, containing 50 μg/ml kanamycin. .. After the initial incubation, the cell culture was diluted into 800 ml LB media with the selection agent and incubated at 37°C until OD600 reached 0.8.

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: .. The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark). .. E. coli cells harboring Ii049 were broken by an ultrasonic disintegrator (Cat. #BILON-650Y, Shanghai Bilon Instrument Co. Ltd., China) in a crushed-ice bath at an output level of 5 and a 50 duty cycle for 1 min with 0.5 s interval every second.

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: These constructs produce recombinant proteins bearing an N-terminal hexahistidine tag that can be removed by cleavage with thrombin, leaving the residual sequence GSH– at the N-termini of the mature proteins. .. Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking.

    Article Title: Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6
    Article Snippet: Paragraph title: Cloning of PSF protein, truncated RBD and DBD protein, expression and purification of recombinant proteins ... The plasmid was transformed into Escherichia coli strain BL21 (Novagen).

    Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells
    Article Snippet: .. Recombinant protein purification The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA). .. Cells were cultured in Luria-Bertani medium and selected with ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml).

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: Paragraph title: Protein recombinant expression and purification ... Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen).

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: Paragraph title: Preparation of recombinant CAMP-hMT1A protein ... The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA).

    Article Title: Curcumin protects mice from Staphylococcus aureus pneumonia by interfering with the self-assembly process of α-hemolysin
    Article Snippet: .. After transforming the recombinant plasmids into Escherichia coli strain BL21(DE3) (Novagen), the soluble Hla proteins were obtained from induced E. coli cells under the appropriate conditions for high-level expression and purification of recombinant Hla protein described by Qiu et al . .. Fluorescence-quenching assay In our paper, fluorescence quenching can be described by the following Scatchard equation: r /D f = nK − rK , where r is the ligand amount of substance per mole of protein binding (r ≈ ΔF /F 0 ), D f is the free concentration of CUR, K is the binding constant and n is the number of binding sites.

    Mutagenesis:

    Article Title: CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis
    Article Snippet: Complementary DNAs encoding wild-type or mutant forms of human p53, each tagged at its N terminus with the HA epitope, were subcloned into pCGN. .. His6 -tagged proteins were expressed in Escherichia coli strain BL21(DE3)pLys(S) (Novagen).

    Article Title: Curcumin protects mice from Staphylococcus aureus pneumonia by interfering with the self-assembly process of α-hemolysin
    Article Snippet: Paragraph title: Mutagenesis of the Hla protein ... After transforming the recombinant plasmids into Escherichia coli strain BL21(DE3) (Novagen), the soluble Hla proteins were obtained from induced E. coli cells under the appropriate conditions for high-level expression and purification of recombinant Hla protein described by Qiu et al .

    Isolation:

    Article Title: Genetically designing a more potent anti-pancreatic cancer agent by simultaneously cotargeting human IL-13 and EGF receptors in a mouse xenograft model
    Article Snippet: Paragraph title: Isolation of inclusion bodies, refolding and purification ... Plasmids were transformed into Escherichia coli strain BL21(DE3) (Novagen, Madison WI).

    Article Title: Heterodimeric bispecific single chain variable fragments (scFv) killer engagers (BiKEs) enhance NK-cell activity against CD133+ colorectal cancer cells
    Article Snippet: Paragraph title: 2.2 Inclusion Body Isolation ... For bacterial protein expression, plasmids were transformed into Escherichia coli strain BL21(DE3) (Novagen, Madison WI).

    Nickel Column:

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark). .. After centrifugation (19,000 × g, 20 min, at 4°CC), the supernatants were applied to a 5 mL nickel column (Bio-Scale Mini Profinity IMAC Cartridges) for Ii049 recombinant protein purification according to the manufacturer's recommendation (Cat. #732-4612, Bio-Rad Laboratories, USA).

    Electrophoretic Mobility Shift Assay:

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: Paragraph title: Electrophoretic mobility shift assay ... The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark).

    Purification:

    Article Title: Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy
    Article Snippet: .. Recombinant proteins purification T7‐His‐tagged ATG16L172–307 and mutants were expressed from pET28a plasmid in Escherichia coli strain BL21 (DE3, Novagen) and grown overnight at 37°C in 50 ml of lysogeny broth (LB) media, containing 50 μg/ml kanamycin. .. After the initial incubation, the cell culture was diluted into 800 ml LB media with the selection agent and incubated at 37°C until OD600 reached 0.8.

    Article Title: Disabling Cas9 by an anti-CRISPR DNA mimic
    Article Snippet: Paragraph title: Protein expression and purification ... The GST-AcrIIA4 fusion protein was overexpressed in Escherichia coli strain BL21 (Novagen) with overnight induction of 0.25 mM isopropyl-β-d -thiogalactopyranoside at 18°C.

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: Paragraph title: 2.1. Protein expression and purification   ... Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking.

    Article Title: Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6
    Article Snippet: Paragraph title: Cloning of PSF protein, truncated RBD and DBD protein, expression and purification of recombinant proteins ... The plasmid was transformed into Escherichia coli strain BL21 (Novagen).

    Article Title: Genetically designing a more potent anti-pancreatic cancer agent by simultaneously cotargeting human IL-13 and EGF receptors in a mouse xenograft model
    Article Snippet: Paragraph title: Isolation of inclusion bodies, refolding and purification ... Plasmids were transformed into Escherichia coli strain BL21(DE3) (Novagen, Madison WI).

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: Paragraph title: Protein recombinant expression and purification ... Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen).

    Article Title: Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery
    Article Snippet: The construct was transformed into E. coli strain BL21 (Novagen), and protein expression was induced by IPTG (1 mM) at 160 overnight. .. GST-His-Tagged fusion protein was purified using Ni-NTA resin (Qiagen) according to manufacturer’s instructions.

    Article Title: Curcumin protects mice from Staphylococcus aureus pneumonia by interfering with the self-assembly process of α-hemolysin
    Article Snippet: .. After transforming the recombinant plasmids into Escherichia coli strain BL21(DE3) (Novagen), the soluble Hla proteins were obtained from induced E. coli cells under the appropriate conditions for high-level expression and purification of recombinant Hla protein described by Qiu et al . .. Fluorescence-quenching assay In our paper, fluorescence quenching can be described by the following Scatchard equation: r /D f = nK − rK , where r is the ligand amount of substance per mole of protein binding (r ≈ ΔF /F 0 ), D f is the free concentration of CUR, K is the binding constant and n is the number of binding sites.

    Article Title: Mechanism of microtubule stabilization by taccalonolide AJ
    Article Snippet: .. Protein expression and purification The stathmin-like domain of RB3 (RB3-SLD) and TTL were overexpressed in Escherichia coli strain BL21(DE3) (Novagen). .. Cells expressing RB3-SLD were collected by centrifugation and resuspended in loading buffer (20 mM Tris-HCl (pH 8.0), 1 mM EGTA, 2 mM dithiothreitol (DTT)) and then lysed by an ultrahigh-pressure homogenizer (JNBIO).

    Protein Purification:

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark). .. After centrifugation (19,000 × g, 20 min, at 4°CC), the supernatants were applied to a 5 mL nickel column (Bio-Scale Mini Profinity IMAC Cartridges) for Ii049 recombinant protein purification according to the manufacturer's recommendation (Cat. #732-4612, Bio-Rad Laboratories, USA).

    Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells
    Article Snippet: .. Recombinant protein purification The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA). .. Cells were cultured in Luria-Bertani medium and selected with ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml).

    Polymerase Chain Reaction:

    Article Title: Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6
    Article Snippet: After purification, PCR product was digested with Nde I and Xho I, then cloned into pBluescript II KS+ to produce pBS-PSF. .. The plasmid was transformed into Escherichia coli strain BL21 (Novagen).

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The Hin dIII-EGFP- Xho I and Nco I-TAT- Hin dIII PCR fragments were cloned into pET28a(+) to prepare the pET28a-TAT-hMT1A and pET28a-CAMP-hMT1A plasmids, respectively. .. The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA).

    Selection:

    Article Title: Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy
    Article Snippet: Recombinant proteins purification T7‐His‐tagged ATG16L172–307 and mutants were expressed from pET28a plasmid in Escherichia coli strain BL21 (DE3, Novagen) and grown overnight at 37°C in 50 ml of lysogeny broth (LB) media, containing 50 μg/ml kanamycin. .. After the initial incubation, the cell culture was diluted into 800 ml LB media with the selection agent and incubated at 37°C until OD600 reached 0.8.

    Activated Clotting Time Assay:

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The sequence of the synthetic gene was CC ATG G GC TAT GGC AGG AAG AAG CGG AGA CAG CGA CGA CGA TTG TTG CGC GCT GCC CTG CGC AAG GCT GCC CTG GGA AAG CTT ATG GAC CCC AAC TGC TCC TGC GCC ACT GGT GGC TCC TGC ACC TGC ACT GGC TCC TGC AAA TGC AAA GAG TGC AAA TGC ACC TCC TGC AAG AAG AGC TGC TGC TCC TGC TGC CCC ATG AGC TGT GCC AAG TGT GCC CAG GGC TGC ATC TGC AAA GGG GCA TCA GAG AAG TGC AGC TGC TGT GCC CTC GAG . .. The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA).

    Plasmid Preparation:

    Article Title: Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy
    Article Snippet: .. Recombinant proteins purification T7‐His‐tagged ATG16L172–307 and mutants were expressed from pET28a plasmid in Escherichia coli strain BL21 (DE3, Novagen) and grown overnight at 37°C in 50 ml of lysogeny broth (LB) media, containing 50 μg/ml kanamycin. .. After the initial incubation, the cell culture was diluted into 800 ml LB media with the selection agent and incubated at 37°C until OD600 reached 0.8.

    Article Title: Engineering of a thermostable viral polymerase using metagenome-derived diversity for highly sensitive and specific RT-PCR
    Article Snippet: .. The DNA constructs were then assembled with a tac promoter-based expression vector and used to transform Escherichia coli strain BL21 (EMD Millipore). .. Magma fusion The 5′→3′ nuclease domain of the Taq polymerase gene (GenBank BAA06775 ) was PCR amplified using Veraseq 2.0 polymerase (QIAGEN) and primers Taqexo-F and Taqexo-R.

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: Electrophoretic mobility shift assay The full length coding sequence of Ii049 was amplified with the primers Ii049 -pET-F and Ii049 -pET-R (Supplementary Table ), and inserted into the expression vector pET32a (Novagen, Denmark) between the Nco I and Hind III sites for the generation of Ii049 recombinant protein. .. The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark).

    Article Title: Disabling Cas9 by an anti-CRISPR DNA mimic
    Article Snippet: Phage-encoded anti-CRISPR AcrIIA4 ( ) was subcloned using Clontech’s In-Fusion cloning system into a pGEX-6P-1 expression vector with a glutathione S -transferase (GST) affinity tag and a PreScission Protease cleavage site at the N terminus. .. The GST-AcrIIA4 fusion protein was overexpressed in Escherichia coli strain BL21 (Novagen) with overnight induction of 0.25 mM isopropyl-β-d -thiogalactopyranoside at 18°C.

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: The genes for Slr0204 and AtDHNAT1 were cloned from their respective organisms into the bacterial expression vector pET15b (Novagen) between the Nde I and Xho I restriction sites. .. Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking.

    Article Title: Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6
    Article Snippet: .. The plasmid was transformed into Escherichia coli strain BL21 (Novagen). .. His-tagged PSF protein was expressed in 200 ml Overnight Express medium (Novagen) for 24h at 25°C.

    Article Title: Scaffold protein FHL2 facilitates MDM2-mediated degradation of IER3 to regulate proliferation of cervical cancer cells
    Article Snippet: .. Recombinant protein purification The pGEX4T1-FHL2 plasmid was expressed in Rosetta 2 (DE3) competent cells, a derivative strain of Escherichia coli strain BL21 (EMD Millipore, Billerica, MA, USA). .. Cells were cultured in Luria-Bertani medium and selected with ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml).

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: .. Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen). ..

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: .. The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA). .. The cells were then cultured in LB medium containing 50 μg/ml ampicillin at 37 °C for 4 h, and CAMP-hMT1A protein expression was induced by adding 1 mM isopropyl-β- d -thio-galactoside and incubating overnight at 26 °C.

    Article Title: Plant AtEH/Pan1 proteins drive autophagosome formation at ER-PM contact sites with actin and endocytic machinery
    Article Snippet: The full-length AtEH1/Pan1 cDNA was cloned into the SacI/XhoI sites of the pET42a vector (with N-terminal GST and HisTag) for protein expression. .. The construct was transformed into E. coli strain BL21 (Novagen), and protein expression was induced by IPTG (1 mM) at 160 overnight.

    Affinity Chromatography:

    Article Title: Disabling Cas9 by an anti-CRISPR DNA mimic
    Article Snippet: The GST-AcrIIA4 fusion protein was overexpressed in Escherichia coli strain BL21 (Novagen) with overnight induction of 0.25 mM isopropyl-β-d -thiogalactopyranoside at 18°C. .. The soluble fractions from the lysates were purified by affinity chromatography using Glutathione Sepharose 4B resin.

    Positron Emission Tomography:

    Article Title: AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes
    Article Snippet: Electrophoretic mobility shift assay The full length coding sequence of Ii049 was amplified with the primers Ii049 -pET-F and Ii049 -pET-R (Supplementary Table ), and inserted into the expression vector pET32a (Novagen, Denmark) between the Nco I and Hind III sites for the generation of Ii049 recombinant protein. .. The resulting Ii049 recombinant protein was expressed in Escherichia coli strain BL21(DE3) (Novagen, Denmark).

    Produced:

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: Protein recombinant expression and purification C. elegans SAS-5 fragments were produced and purified as described previously ( ). .. Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen).

    FLAG-tag:

    Article Title: CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis
    Article Snippet: Complementary DNAs encoding mouse histone H1 variants, each tagged at its N terminus with three copies of the Flag epitope, were subcloned into pcDNA3. .. His6 -tagged proteins were expressed in Escherichia coli strain BL21(DE3)pLys(S) (Novagen).

    CTG Assay:

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The sequence of the synthetic gene was CC ATG G GC TAT GGC AGG AAG AAG CGG AGA CAG CGA CGA CGA TTG TTG CGC GCT GCC CTG CGC AAG GCT GCC CTG GGA AAG CTT ATG GAC CCC AAC TGC TCC TGC GCC ACT GGT GGC TCC TGC ACC TGC ACT GGC TCC TGC AAA TGC AAA GAG TGC AAA TGC ACC TCC TGC AAG AAG AGC TGC TGC TCC TGC TGC CCC ATG AGC TGT GCC AAG TGT GCC CAG GGC TGC ATC TGC AAA GGG GCA TCA GAG AAG TGC AGC TGC TGT GCC CTC GAG . .. The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA).

    Lysis:

    Article Title: Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy
    Article Snippet: Recombinant proteins purification T7‐His‐tagged ATG16L172–307 and mutants were expressed from pET28a plasmid in Escherichia coli strain BL21 (DE3, Novagen) and grown overnight at 37°C in 50 ml of lysogeny broth (LB) media, containing 50 μg/ml kanamycin. .. Cell lysis was performed as previously described (Archna & Scrima, ).

    Article Title: Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis
    Article Snippet: Proteins were expressed in Escherichia coli strain BL21(DE3) (EMD Millipore) by growing cells to mid-log phase (OD600 = 0.4–0.6) in LB medium supplemented with 100 µg ml−1 ampicillin at 310 K with shaking. .. Cell pellets were thawed on ice and resuspended in lysis buffer (50 m M HEPES pH 7.5, 300 m M NaCl, 10 m M imidazole) at a 5:1 volume:mass ratio of buffer to cell pellet.

    Article Title: Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson’s disease models
    Article Snippet: The pET28a-CAMP-hMT1A plasmid was transformed into Escherichia coli strain BL21(DE3)pLysS (Novagen, Madison, WI, USA). .. Cell pellets were collected by centrifugation and suspended in lysis buffer (300 mM KCl, 49 mM KH2 PO4 , and 4.9 mM imidazole) in the presence of 100 mM phenylmethylsulfonyl fluoride (PMSF), followed by sonication for eight cycles of 30 s each .

    Variant Assay:

    Article Title: Interaction between the Caenorhabditis elegans centriolar protein SAS-5 and microtubules facilitates organelle assembly
    Article Snippet: .. Briefly, gene fragments encoding SAS-5 WT or variant constructs were cloned in a pFloat vector with a human rhinovirus (HRV) 3C protease-cleavable N-terminal hexahistidine-SUMO tag, and transformed into Escherichia coli strain BL21(DE3) Rosetta2 (Novagen). ..

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