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    Structured Review

    Thermo Fisher e coli bl21
    AP2µ Binds to Isolated CD79a but not CD79b. Panel A , Amino acid sequences of CD79a and CD79b cytoplasmic domains. YxxØ putative AP2 binding motifs underlined. Panel B , AP2µ expressed as a Gal4 activation domain fusion protein was assayed for specific interaction with the cytoplasmic domain of either CD79a or CD79b fused to the Gal4 DNA binding domain. Growth on histidine deficient (His-) plates indicates an AP2–CD79 interaction. The cytoplasmic domain of TGN38 contains a known AP2 binding YxxØ motif and served as a positive control, while the cytoplasmic domain of OCA2 contains a dileucine motif (which does not bind AP2µ) and served as a negative control. Data are representative of 2 experiments. Panel C , Diagram of the GST-CD79 cytoplasmic domain–AP2µ direct binding assay. Panel D , The cytoplasmic domains of CD79a and CD79b were expressed as GST fusion proteins in <t>BL21</t> E. coli cells. GST-fusion proteins were captured from cell lysates on glutathione beads and the resulting matrix was tested for binding to in vitro translated, biotin-labeled AP2µ. The AP2 binding motif from TGN38, (SDYQRL) 3 , and a non-AP2-binding derivation containing a tyrosine to glycine substitution, (SDGQRL) 3 , fused to GST served as positive and negative controls, respectively. Binding is expressed as a percentage of (SDYQRL) 3 –AP2 interactions. Data is the mean of 3 independent experiments ± S.E.M. Statistical comparisons were measured between SDYQRL and other samples.
    E Coli Bl21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Cis and Trans Regulatory Mechanisms Control AP2-Mediated B Cell Receptor Endocytosis via Select Tyrosine-Based Motifs"

    Article Title: Cis and Trans Regulatory Mechanisms Control AP2-Mediated B Cell Receptor Endocytosis via Select Tyrosine-Based Motifs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054938

    AP2µ Binds to Isolated CD79a but not CD79b. Panel A , Amino acid sequences of CD79a and CD79b cytoplasmic domains. YxxØ putative AP2 binding motifs underlined. Panel B , AP2µ expressed as a Gal4 activation domain fusion protein was assayed for specific interaction with the cytoplasmic domain of either CD79a or CD79b fused to the Gal4 DNA binding domain. Growth on histidine deficient (His-) plates indicates an AP2–CD79 interaction. The cytoplasmic domain of TGN38 contains a known AP2 binding YxxØ motif and served as a positive control, while the cytoplasmic domain of OCA2 contains a dileucine motif (which does not bind AP2µ) and served as a negative control. Data are representative of 2 experiments. Panel C , Diagram of the GST-CD79 cytoplasmic domain–AP2µ direct binding assay. Panel D , The cytoplasmic domains of CD79a and CD79b were expressed as GST fusion proteins in BL21 E. coli cells. GST-fusion proteins were captured from cell lysates on glutathione beads and the resulting matrix was tested for binding to in vitro translated, biotin-labeled AP2µ. The AP2 binding motif from TGN38, (SDYQRL) 3 , and a non-AP2-binding derivation containing a tyrosine to glycine substitution, (SDGQRL) 3 , fused to GST served as positive and negative controls, respectively. Binding is expressed as a percentage of (SDYQRL) 3 –AP2 interactions. Data is the mean of 3 independent experiments ± S.E.M. Statistical comparisons were measured between SDYQRL and other samples.
    Figure Legend Snippet: AP2µ Binds to Isolated CD79a but not CD79b. Panel A , Amino acid sequences of CD79a and CD79b cytoplasmic domains. YxxØ putative AP2 binding motifs underlined. Panel B , AP2µ expressed as a Gal4 activation domain fusion protein was assayed for specific interaction with the cytoplasmic domain of either CD79a or CD79b fused to the Gal4 DNA binding domain. Growth on histidine deficient (His-) plates indicates an AP2–CD79 interaction. The cytoplasmic domain of TGN38 contains a known AP2 binding YxxØ motif and served as a positive control, while the cytoplasmic domain of OCA2 contains a dileucine motif (which does not bind AP2µ) and served as a negative control. Data are representative of 2 experiments. Panel C , Diagram of the GST-CD79 cytoplasmic domain–AP2µ direct binding assay. Panel D , The cytoplasmic domains of CD79a and CD79b were expressed as GST fusion proteins in BL21 E. coli cells. GST-fusion proteins were captured from cell lysates on glutathione beads and the resulting matrix was tested for binding to in vitro translated, biotin-labeled AP2µ. The AP2 binding motif from TGN38, (SDYQRL) 3 , and a non-AP2-binding derivation containing a tyrosine to glycine substitution, (SDGQRL) 3 , fused to GST served as positive and negative controls, respectively. Binding is expressed as a percentage of (SDYQRL) 3 –AP2 interactions. Data is the mean of 3 independent experiments ± S.E.M. Statistical comparisons were measured between SDYQRL and other samples.

    Techniques Used: Isolation, Binding Assay, Activation Assay, Positive Control, Negative Control, In Vitro, Labeling

    2) Product Images from "The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection"

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.810440

    Recombinant enolase1 from C. albicans has TGase activity. The C. albicans ENO1 gene was cloned in the pCold II plasmid and transformed into E. coli BL21 (DE3) pLysS-competent cells; protein production was induced at 23 °C for 24 h. A , Eno1 protein was purified by IMAC with a Ni 2+ -NTA–agarose column in native conditions as described, and elution fractions were evaluated by 12% SDS-PAGE; MWM , protein molecular weight markers. Empty vector was also transformed in bacteria and passed through the same IMAC column, and the fractions obtained were also analyzed as a negative control (data not shown). B , Western blot of purified recombinant protein using anti-His–tag polyclonal antibodies ( lane 2 ) and rabbit anti-rCaEno1 protein ( lane 3 ). C , Western blot of C. albicans cell fractions using anti-rCaEno1 polyclonal antibodies. WPE , whole-protein extracts; CW , cell wall fraction; MMF , mixed membrane fraction; S-35K , soluble cytosolic fraction. Arrows indicate Eno1 protein. D , enolase activity was determined with purified rCaEno1 protein. E , TGase activity determined with purified rCaEno1 protein. These results allowed us to conclude that rCaEno1 protein has both enolase and transglutaminase activities. Statistical unpaired t test. *, p
    Figure Legend Snippet: Recombinant enolase1 from C. albicans has TGase activity. The C. albicans ENO1 gene was cloned in the pCold II plasmid and transformed into E. coli BL21 (DE3) pLysS-competent cells; protein production was induced at 23 °C for 24 h. A , Eno1 protein was purified by IMAC with a Ni 2+ -NTA–agarose column in native conditions as described, and elution fractions were evaluated by 12% SDS-PAGE; MWM , protein molecular weight markers. Empty vector was also transformed in bacteria and passed through the same IMAC column, and the fractions obtained were also analyzed as a negative control (data not shown). B , Western blot of purified recombinant protein using anti-His–tag polyclonal antibodies ( lane 2 ) and rabbit anti-rCaEno1 protein ( lane 3 ). C , Western blot of C. albicans cell fractions using anti-rCaEno1 polyclonal antibodies. WPE , whole-protein extracts; CW , cell wall fraction; MMF , mixed membrane fraction; S-35K , soluble cytosolic fraction. Arrows indicate Eno1 protein. D , enolase activity was determined with purified rCaEno1 protein. E , TGase activity determined with purified rCaEno1 protein. These results allowed us to conclude that rCaEno1 protein has both enolase and transglutaminase activities. Statistical unpaired t test. *, p

    Techniques Used: Recombinant, Activity Assay, Clone Assay, Plasmid Preparation, Transformation Assay, Purification, SDS Page, Molecular Weight, Negative Control, Western Blot

    3) Product Images from "The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection"

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.810440

    Recombinant enolase1 from C. albicans has TGase activity. The C. albicans ENO1 gene was cloned in the pCold II plasmid and transformed into E. coli BL21 (DE3) pLysS-competent cells; protein production was induced at 23 °C for 24 h. A , Eno1 protein was purified by IMAC with a Ni 2+ -NTA–agarose column in native conditions as described, and elution fractions were evaluated by 12% SDS-PAGE; MWM , protein molecular weight markers. Empty vector was also transformed in bacteria and passed through the same IMAC column, and the fractions obtained were also analyzed as a negative control (data not shown). B , Western blot of purified recombinant protein using anti-His–tag polyclonal antibodies ( lane 2 ) and rabbit anti-rCaEno1 protein ( lane 3 ). C , Western blot of C. albicans cell fractions using anti-rCaEno1 polyclonal antibodies. WPE , whole-protein extracts; CW , cell wall fraction; MMF , mixed membrane fraction; S-35K , soluble cytosolic fraction. Arrows indicate Eno1 protein. D , enolase activity was determined with purified rCaEno1 protein. E , TGase activity determined with purified rCaEno1 protein. These results allowed us to conclude that rCaEno1 protein has both enolase and transglutaminase activities. Statistical unpaired t test. *, p
    Figure Legend Snippet: Recombinant enolase1 from C. albicans has TGase activity. The C. albicans ENO1 gene was cloned in the pCold II plasmid and transformed into E. coli BL21 (DE3) pLysS-competent cells; protein production was induced at 23 °C for 24 h. A , Eno1 protein was purified by IMAC with a Ni 2+ -NTA–agarose column in native conditions as described, and elution fractions were evaluated by 12% SDS-PAGE; MWM , protein molecular weight markers. Empty vector was also transformed in bacteria and passed through the same IMAC column, and the fractions obtained were also analyzed as a negative control (data not shown). B , Western blot of purified recombinant protein using anti-His–tag polyclonal antibodies ( lane 2 ) and rabbit anti-rCaEno1 protein ( lane 3 ). C , Western blot of C. albicans cell fractions using anti-rCaEno1 polyclonal antibodies. WPE , whole-protein extracts; CW , cell wall fraction; MMF , mixed membrane fraction; S-35K , soluble cytosolic fraction. Arrows indicate Eno1 protein. D , enolase activity was determined with purified rCaEno1 protein. E , TGase activity determined with purified rCaEno1 protein. These results allowed us to conclude that rCaEno1 protein has both enolase and transglutaminase activities. Statistical unpaired t test. *, p

    Techniques Used: Recombinant, Activity Assay, Clone Assay, Plasmid Preparation, Transformation Assay, Purification, SDS Page, Molecular Weight, Negative Control, Western Blot

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    Clone Assay:

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    Centrifugation:

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    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
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    Amplification:

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    Filtration:

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    Synthesized:

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    Construct:

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    Article Title: Development and application of a transcriptional sensor for detection of heterologous acrylic acid production in E. coli
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    Incubation:

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    Article Title: The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading
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    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells. .. Cells were incubated at 4 °C for 1 h and sonicated three times in ice for 10 s at 60% amplitude in a CPX 130PB Ultrasonic Processor (Cole-Parmer, Verno Hills, IL) with cooling for 1 min in ice between each sonication cycle, and the cell-free extract was obtained by centrifugation at 10,000 × g for 30 min at 4 °C.

    Article Title: Cis and Trans Regulatory Mechanisms Control AP2-Mediated B Cell Receptor Endocytosis via Select Tyrosine-Based Motifs
    Article Snippet: E. coli BL21 (Invitrogen, Grand Island, NY, USA) expressing the GST-fusion proteins were grown and induced with IPTG according to the manufacturer’s protocol. .. GST-fusion proteins were bound to glutathione beads, washed and incubated with biotinylated AP2µ (AP2µ-btn) for 30 minutes.

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells. .. Cells were incubated at 4 °C for 1 h and sonicated three times in ice for 10 s at 60% amplitude in a CPX 130PB Ultrasonic Processor (Cole-Parmer, Verno Hills, IL) with cooling for 1 min in ice between each sonication cycle, and the cell-free extract was obtained by centrifugation at 10,000 × g for 30 min at 4 °C.

    Activity Assay:

    Article Title: Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme
    Article Snippet: .. Gene expression and protein production and purification For enzyme activity assays, pBS12067 was transformed into E. coli BL21(DE3) (Life Technologies) and grown in 1 L of lysogeny broth (LB) at 37 °C with shaking at 250 rpm until an OD600 of 0.6 was reached. .. The culture was cooled to 4 °C, gene expression was induced with the addition of 0.25 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and the cells were grown overnight at 18 °C with shaking.

    Expressing:

    Article Title: S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 [OPEN]
    Article Snippet: Paragraph title: Protein Expression and Purification ... The pET28a-S5H construct was introduced into Escherichia coli BL21 (DE3, pLys3; Invitrogen).

    Article Title: Development and application of a transcriptional sensor for detection of heterologous acrylic acid production in E. coli
    Article Snippet: .. Expression and purification of RAPc8 amidase variants The RAPc8 amidase constructs were cloned with a C-terminal 6xHIS tag and transformed into Escherichia coli BL21(DE3) (Invitrogen) competent cells. ..

    Article Title: Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme
    Article Snippet: .. Gene expression and protein production and purification For enzyme activity assays, pBS12067 was transformed into E. coli BL21(DE3) (Life Technologies) and grown in 1 L of lysogeny broth (LB) at 37 °C with shaking at 250 rpm until an OD600 of 0.6 was reached. .. The culture was cooled to 4 °C, gene expression was induced with the addition of 0.25 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and the cells were grown overnight at 18 °C with shaking.

    Article Title: Mycophenolic Acid Derivatives with Immunosuppressive Activity from the Coral-Derived Fungus Penicillium bialowiezense
    Article Snippet: Paragraph title: 3.4.1. IMPDH2 Expression and Purification ... After the recombinant plasmids were verified by sequencing, the plasmid was transformed into Escherichia coli BL21(DE3) (Invitrogen), which was grown in LB medium at 37 °C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) and grown at 20 °C for 16 h. The cell pellet was harvested and re-suspended in 30 mL buffer (20 mM Tris pH 8.5, 200 mM NaCl, and 10 mM imidazole), followed by disruption on a French press.

    Article Title: Prime-boost and recombinant protein vaccination strategies using Sm-p80 protects against Schistosoma mansoni infection in the mouse model to levels previously attainable only by the irradiated cercarial vaccine
    Article Snippet: .. Escherichia coli BL21 (DE3; Invitrogen Corp., Carlsbad, CA, USA) were used as the expression host strain. .. The expressed proteins were initially purified via Ni-nitrilotriacetic acid-agarose resin (Qiagen Inc., Valencia, CA, USA), followed by gel filtration chromatography using a Sephadex G-150 column.

    Article Title: The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading
    Article Snippet: Pex1/Pex6 purification Pex1-FLAG and His-Pex6 wild type and mutant complexes were co-expressed in E. coli BL21* (ThermoFisher Scientific) from the pETDuet and pCOLADuet vectors. .. The expression strain was grown in 6 L of DYT (16 g tryptone, 10 g yeast extract, 5 g NaCl) and appropriate antibiotics at 30 °C and induced at OD600 = 0.6–0.9 with IPTG (final concentration C f =0.3 mM) before overnight incubation at 18 °C.

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: Paragraph title: Expression of recombinant C. albicans Eno1 protein and generation of anti-rCaEno1 polyclonal antibodies ... It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells.

    Article Title: Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet
    Article Snippet: Paragraph title: Identification of GG1 based on amino acid sequence data, cloning, and expression of rGG1 ... The pET-GG1 construct was transformed into E coli BL21 (DE3; Invitrogen, Carlsbad, Calif), and rGG1 was expressed in liquid cultures and purified, as previously described.

    Article Title: Human C1q Induces Apoptosis in an Ovarian Cancer Cell Line via Tumor Necrosis Factor Pathway
    Article Snippet: .. Expression constructs pKBM-A, pKBM-B, and pKBM-C were transformed into E. coli BL21 (Invitrogen) cells in the presence of ampicillin (100 µg/ml). ..

    Article Title: Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on polymer chip
    Article Snippet: Paragraph title: Construction, expression and purification of stx2 ZFPs ... Proteins were expressed in E. coli BL21 (Invitrogen) upon induction with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.6–0.8 for 3 h at 37°C.

    Article Title: Cis and Trans Regulatory Mechanisms Control AP2-Mediated B Cell Receptor Endocytosis via Select Tyrosine-Based Motifs
    Article Snippet: .. E. coli BL21 (Invitrogen, Grand Island, NY, USA) expressing the GST-fusion proteins were grown and induced with IPTG according to the manufacturer’s protocol. .. The bacteria were lysed by sonication in a buffer containing 0.5% NP-40, 20mM Tris pH 8.0, 100mM NaCl and 1mM EDTA.

    Article Title: Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y
    Article Snippet: Paragraph title: Expression and purification of rMHJ_0659 and rMHJ_0522 ... The recombinant construct was transformed into E. coli BL21 (Invitrogen) as per the manufacturer instructions and grown overnight in LB supplemented with 100 mg/mL ampicillin.

    Bradford Assay:

    Article Title: Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on polymer chip
    Article Snippet: Proteins were expressed in E. coli BL21 (Invitrogen) upon induction with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.6–0.8 for 3 h at 37°C. .. Concentration and purity were assessed by Coomassi-stained polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS–PAGE) and Bradford assay using bovine serum albumen (BSA) standards.

    Transformation Assay:

    Article Title: LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain
    Article Snippet: .. The cloned plasmid was transformed into E. coli BL21(DE3) (Invitrogen, Carlsbad, CA, USA). ..

    Article Title: Development and application of a transcriptional sensor for detection of heterologous acrylic acid production in E. coli
    Article Snippet: .. Expression and purification of RAPc8 amidase variants The RAPc8 amidase constructs were cloned with a C-terminal 6xHIS tag and transformed into Escherichia coli BL21(DE3) (Invitrogen) competent cells. ..

    Article Title: Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme
    Article Snippet: .. Gene expression and protein production and purification For enzyme activity assays, pBS12067 was transformed into E. coli BL21(DE3) (Life Technologies) and grown in 1 L of lysogeny broth (LB) at 37 °C with shaking at 250 rpm until an OD600 of 0.6 was reached. .. The culture was cooled to 4 °C, gene expression was induced with the addition of 0.25 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and the cells were grown overnight at 18 °C with shaking.

    Article Title: Mycophenolic Acid Derivatives with Immunosuppressive Activity from the Coral-Derived Fungus Penicillium bialowiezense
    Article Snippet: .. After the recombinant plasmids were verified by sequencing, the plasmid was transformed into Escherichia coli BL21(DE3) (Invitrogen), which was grown in LB medium at 37 °C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) and grown at 20 °C for 16 h. The cell pellet was harvested and re-suspended in 30 mL buffer (20 mM Tris pH 8.5, 200 mM NaCl, and 10 mM imidazole), followed by disruption on a French press. .. Cell debris was removed by centrifugation at 21,000 rpm for 30 min.

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: .. It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells. .. The induction of recombinant CaEno1 protein (rCaEno1) was achieved with 0.5 mm isopropyl β-d -thiogalactopyranoside for 24 h at 23 °C in Luria-Bertani medium at 200 rpm.

    Article Title: Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet
    Article Snippet: .. The pET-GG1 construct was transformed into E coli BL21 (DE3; Invitrogen, Carlsbad, Calif), and rGG1 was expressed in liquid cultures and purified, as previously described. .. Multiple sequence alignment of GG1 with homologous proteins from other cereals An NCBI Protein blast ( http://blast.ncbi.nlm.nih.gov ) search of GG1 (SwissProt ID: P08453.1) against spelt (Triticum spelta) , rye (Secale cereale) , barley (Hordeum vulgare L) , maize (Zea mays) , rice (Oryza sativa) , and oat (Avena sativa) was performed.

    Article Title: Human C1q Induces Apoptosis in an Ovarian Cancer Cell Line via Tumor Necrosis Factor Pathway
    Article Snippet: .. Expression constructs pKBM-A, pKBM-B, and pKBM-C were transformed into E. coli BL21 (Invitrogen) cells in the presence of ampicillin (100 µg/ml). ..

    Article Title: Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y
    Article Snippet: .. The recombinant construct was transformed into E. coli BL21 (Invitrogen) as per the manufacturer instructions and grown overnight in LB supplemented with 100 mg/mL ampicillin. .. LB supplemented with 100 mg/mL ampicillin was inoculated with overnight transformation culture and grown until mid-log growth phase.

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: .. It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells. .. The induction of recombinant CaEno1 protein (rCaEno1) was achieved with 0.5 m m isopropyl β- d -thiogalactopyranoside for 24 h at 23 °C in Luria-Bertani medium at 200 rpm.

    Chromatography:

    Article Title: Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme
    Article Snippet: Gene expression and protein production and purification For enzyme activity assays, pBS12067 was transformed into E. coli BL21(DE3) (Life Technologies) and grown in 1 L of lysogeny broth (LB) at 37 °C with shaking at 250 rpm until an OD600 of 0.6 was reached. .. The supernatant containing PtmA2 was purified by nickel-affinity chromatography using an ÄKTA FPLC system (GE Healthcare Biosciences) equipped with a HisTrap column.

    Article Title: Prime-boost and recombinant protein vaccination strategies using Sm-p80 protects against Schistosoma mansoni infection in the mouse model to levels previously attainable only by the irradiated cercarial vaccine
    Article Snippet: Escherichia coli BL21 (DE3; Invitrogen Corp., Carlsbad, CA, USA) were used as the expression host strain. .. The expressed proteins were initially purified via Ni-nitrilotriacetic acid-agarose resin (Qiagen Inc., Valencia, CA, USA), followed by gel filtration chromatography using a Sephadex G-150 column.

    Generated:

    Article Title: LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain
    Article Snippet: The site-directed mutants of LysPBC2 were generated using overlapping PCR with the primers listed in . .. The cloned plasmid was transformed into E. coli BL21(DE3) (Invitrogen, Carlsbad, CA, USA).

    Article Title: Cis and Trans Regulatory Mechanisms Control AP2-Mediated B Cell Receptor Endocytosis via Select Tyrosine-Based Motifs
    Article Snippet: AP2 Binding Assay Biotinylated AP2µ subunit was generated using the TnT Transcription/Translation System combined with the Transcend Biotinylated tRNA (Promega, Madison, WI, USA). .. E. coli BL21 (Invitrogen, Grand Island, NY, USA) expressing the GST-fusion proteins were grown and induced with IPTG according to the manufacturer’s protocol.

    Polymerase Chain Reaction:

    Article Title: LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain
    Article Snippet: The site-directed mutants of LysPBC2 were generated using overlapping PCR with the primers listed in . .. The cloned plasmid was transformed into E. coli BL21(DE3) (Invitrogen, Carlsbad, CA, USA).

    Article Title: S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 [OPEN]
    Article Snippet: The S5H/DMR6 coding sequence was PCR amplified using a pair of primers, S5H_ Bam HI (5′-TTTAAGGATCCATGGCGGCAAAGCTGATATC-3′) and S5H_ Sal I (5′-CATGGTCGACTTAGTTGTTTAGAAAATTCTCGA-3′), and cloned into pET28a (Novagen) to form pET28a-S5H and produce the His-tagged recombinant S5H/DMR6 protein. .. The pET28a-S5H construct was introduced into Escherichia coli BL21 (DE3, pLys3; Invitrogen).

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: Expression of recombinant C. albicans Eno1 protein and generation of anti-rCaEno1 polyclonal antibodies Full-length CaENO1 gene (1323 bp) flanked by KpnI and BamHI restriction sites was amplified by PCR using genomic DNA using the following primers, CaENO1pColdII_Fw(5′-CGGGGTACCATGTCTTACGCCACTAAAATCCA-3′) and CaENO1pColdII_Rv(5′-CGCGGATCCTTACAATTGAGAAGCCTTTTGGAA-3′), and cloned into the pJET1.2/blunt vector (Thermo Fisher Scientific). .. It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells.

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: Full-length CaENO1 gene (1323 bp) flanked by KpnI and BamHI restriction sites was amplified by PCR using genomic DNA using the following primers, CaENO1pColdII_Fw(5′-CGGGGTACCATGTCTTACGCCACTAAAATCCA-3′) and CaENO1pColdII_Rv(5′-CGCGGATCCTTACAATTGAGAAGCCTTTTGGAA-3′), and cloned into the pJET1.2/blunt vector (Thermo Fisher Scientific). .. It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells.

    Sonication:

    Article Title: LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain
    Article Snippet: The cloned plasmid was transformed into E. coli BL21(DE3) (Invitrogen, Carlsbad, CA, USA). .. After thawing, the cells were resuspended in a lysis buffer containing 200 mM NaCl and 50 mM Tris-Cl (pH 8.0) and disrupted by sonication (Sonifier 250; Branson, Danbury, CT, USA).

    Article Title: Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme
    Article Snippet: Gene expression and protein production and purification For enzyme activity assays, pBS12067 was transformed into E. coli BL21(DE3) (Life Technologies) and grown in 1 L of lysogeny broth (LB) at 37 °C with shaking at 250 rpm until an OD600 of 0.6 was reached. .. After harvesting the cells by centrifugation at 4000 g for 15 min at 4 °C, the pellet was resuspended in lysis buffer (100 mM Tris, pH 8.0, containing 300 mM NaCl, 15 mM imidazole, and 10% glycerol), lysed by sonication, and centrifuged at 15,000 g for 20 min at 4 °C.

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells. .. Cells were incubated at 4 °C for 1 h and sonicated three times in ice for 10 s at 60% amplitude in a CPX 130PB Ultrasonic Processor (Cole-Parmer, Verno Hills, IL) with cooling for 1 min in ice between each sonication cycle, and the cell-free extract was obtained by centrifugation at 10,000 × g for 30 min at 4 °C.

    Article Title: Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on polymer chip
    Article Snippet: Proteins were expressed in E. coli BL21 (Invitrogen) upon induction with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.6–0.8 for 3 h at 37°C. .. After sonication, proteins in cell lysates were applied to an amylose resin column (Bio-rad) pre-equilibrated with ZBA + 5 mM DTT, washed with ZBA + 2 M NaCl and ZBA + 1 mM Tris(2-carboxyethyl)phosphine (TCEP), and eluted in ZBA + 10 mM Maltose + 1 mM TCEP.

    Article Title: Cis and Trans Regulatory Mechanisms Control AP2-Mediated B Cell Receptor Endocytosis via Select Tyrosine-Based Motifs
    Article Snippet: E. coli BL21 (Invitrogen, Grand Island, NY, USA) expressing the GST-fusion proteins were grown and induced with IPTG according to the manufacturer’s protocol. .. The bacteria were lysed by sonication in a buffer containing 0.5% NP-40, 20mM Tris pH 8.0, 100mM NaCl and 1mM EDTA.

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells. .. Cells were incubated at 4 °C for 1 h and sonicated three times in ice for 10 s at 60% amplitude in a CPX 130PB Ultrasonic Processor (Cole-Parmer, Verno Hills, IL) with cooling for 1 min in ice between each sonication cycle, and the cell-free extract was obtained by centrifugation at 10,000 × g for 30 min at 4 °C.

    Binding Assay:

    Article Title: Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on polymer chip
    Article Snippet: The pMAL vector was used for bacterial expression of the proteins as fusions with an N-terminal maltose binding protein (MBP) as a purification tag. .. Proteins were expressed in E. coli BL21 (Invitrogen) upon induction with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.6–0.8 for 3 h at 37°C.

    Article Title: Cis and Trans Regulatory Mechanisms Control AP2-Mediated B Cell Receptor Endocytosis via Select Tyrosine-Based Motifs
    Article Snippet: Paragraph title: AP2 Binding Assay ... E. coli BL21 (Invitrogen, Grand Island, NY, USA) expressing the GST-fusion proteins were grown and induced with IPTG according to the manufacturer’s protocol.

    Molecular Weight:

    Article Title: Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet
    Article Snippet: When we searched the SwissProt database for wheat sequences coding for proteins of the corresponding molecular weight containing both peptides, we identified GG1 from Triticum aestivum (SwissProt accession no. P08453.1) as a candidate antigen. .. The pET-GG1 construct was transformed into E coli BL21 (DE3; Invitrogen, Carlsbad, Calif), and rGG1 was expressed in liquid cultures and purified, as previously described.

    Mutagenesis:

    Article Title: The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading
    Article Snippet: .. Pex1/Pex6 purification Pex1-FLAG and His-Pex6 wild type and mutant complexes were co-expressed in E. coli BL21* (ThermoFisher Scientific) from the pETDuet and pCOLADuet vectors. .. The expression strain was grown in 6 L of DYT (16 g tryptone, 10 g yeast extract, 5 g NaCl) and appropriate antibiotics at 30 °C and induced at OD600 = 0.6–0.9 with IPTG (final concentration C f =0.3 mM) before overnight incubation at 18 °C.

    Size-exclusion Chromatography:

    Article Title: Mycophenolic Acid Derivatives with Immunosuppressive Activity from the Coral-Derived Fungus Penicillium bialowiezense
    Article Snippet: After the recombinant plasmids were verified by sequencing, the plasmid was transformed into Escherichia coli BL21(DE3) (Invitrogen), which was grown in LB medium at 37 °C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) and grown at 20 °C for 16 h. The cell pellet was harvested and re-suspended in 30 mL buffer (20 mM Tris pH 8.5, 200 mM NaCl, and 10 mM imidazole), followed by disruption on a French press. .. The protein was further purified with size exclusion chromatography at 20 mM Tris pH 8.5 and 200 mM NaCl.

    Purification:

    Article Title: S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 [OPEN]
    Article Snippet: Paragraph title: Protein Expression and Purification ... The pET28a-S5H construct was introduced into Escherichia coli BL21 (DE3, pLys3; Invitrogen).

    Article Title: Development and application of a transcriptional sensor for detection of heterologous acrylic acid production in E. coli
    Article Snippet: .. Expression and purification of RAPc8 amidase variants The RAPc8 amidase constructs were cloned with a C-terminal 6xHIS tag and transformed into Escherichia coli BL21(DE3) (Invitrogen) competent cells. ..

    Article Title: IscR Regulation of Type 3 Fimbriae Expression in Klebsiella pneumoniae CG43
    Article Snippet: .. Purification of IscR::His6 and IscR3CA ::His6 The plasmids, pET30b-IscR and pET30b-IscR3CA , in E. coli BL21(DE3)[pLysS] (Invitrogen, United States) was used to overexpress the recombinant proteins IscR::His6 and IscR3CA ::His6 , respectively. ..

    Article Title: Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme
    Article Snippet: .. Gene expression and protein production and purification For enzyme activity assays, pBS12067 was transformed into E. coli BL21(DE3) (Life Technologies) and grown in 1 L of lysogeny broth (LB) at 37 °C with shaking at 250 rpm until an OD600 of 0.6 was reached. .. The culture was cooled to 4 °C, gene expression was induced with the addition of 0.25 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), and the cells were grown overnight at 18 °C with shaking.

    Article Title: Mycophenolic Acid Derivatives with Immunosuppressive Activity from the Coral-Derived Fungus Penicillium bialowiezense
    Article Snippet: Paragraph title: 3.4.1. IMPDH2 Expression and Purification ... After the recombinant plasmids were verified by sequencing, the plasmid was transformed into Escherichia coli BL21(DE3) (Invitrogen), which was grown in LB medium at 37 °C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) and grown at 20 °C for 16 h. The cell pellet was harvested and re-suspended in 30 mL buffer (20 mM Tris pH 8.5, 200 mM NaCl, and 10 mM imidazole), followed by disruption on a French press.

    Article Title: Prime-boost and recombinant protein vaccination strategies using Sm-p80 protects against Schistosoma mansoni infection in the mouse model to levels previously attainable only by the irradiated cercarial vaccine
    Article Snippet: Paragraph title: Cloning, expression, and purification of Sm-p80 ... Escherichia coli BL21 (DE3; Invitrogen Corp., Carlsbad, CA, USA) were used as the expression host strain.

    Article Title: The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading
    Article Snippet: .. Pex1/Pex6 purification Pex1-FLAG and His-Pex6 wild type and mutant complexes were co-expressed in E. coli BL21* (ThermoFisher Scientific) from the pETDuet and pCOLADuet vectors. .. The expression strain was grown in 6 L of DYT (16 g tryptone, 10 g yeast extract, 5 g NaCl) and appropriate antibiotics at 30 °C and induced at OD600 = 0.6–0.9 with IPTG (final concentration C f =0.3 mM) before overnight incubation at 18 °C.

    Article Title: Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet
    Article Snippet: .. The pET-GG1 construct was transformed into E coli BL21 (DE3; Invitrogen, Carlsbad, Calif), and rGG1 was expressed in liquid cultures and purified, as previously described. .. Multiple sequence alignment of GG1 with homologous proteins from other cereals An NCBI Protein blast ( http://blast.ncbi.nlm.nih.gov ) search of GG1 (SwissProt ID: P08453.1) against spelt (Triticum spelta) , rye (Secale cereale) , barley (Hordeum vulgare L) , maize (Zea mays) , rice (Oryza sativa) , and oat (Avena sativa) was performed.

    Article Title: Human C1q Induces Apoptosis in an Ovarian Cancer Cell Line via Tumor Necrosis Factor Pathway
    Article Snippet: Paragraph title: Recombinant Expression and Purification of ghA, ghB, and ghC Modules of Human C1q ... Expression constructs pKBM-A, pKBM-B, and pKBM-C were transformed into E. coli BL21 (Invitrogen) cells in the presence of ampicillin (100 µg/ml).

    Article Title: Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on polymer chip
    Article Snippet: Paragraph title: Construction, expression and purification of stx2 ZFPs ... Proteins were expressed in E. coli BL21 (Invitrogen) upon induction with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.6–0.8 for 3 h at 37°C.

    Article Title: Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y
    Article Snippet: Paragraph title: Expression and purification of rMHJ_0659 and rMHJ_0522 ... The recombinant construct was transformed into E. coli BL21 (Invitrogen) as per the manufacturer instructions and grown overnight in LB supplemented with 100 mg/mL ampicillin.

    Sequencing:

    Article Title: S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 [OPEN]
    Article Snippet: The S5H/DMR6 coding sequence was PCR amplified using a pair of primers, S5H_ Bam HI (5′-TTTAAGGATCCATGGCGGCAAAGCTGATATC-3′) and S5H_ Sal I (5′-CATGGTCGACTTAGTTGTTTAGAAAATTCTCGA-3′), and cloned into pET28a (Novagen) to form pET28a-S5H and produce the His-tagged recombinant S5H/DMR6 protein. .. The pET28a-S5H construct was introduced into Escherichia coli BL21 (DE3, pLys3; Invitrogen).

    Article Title: Mycophenolic Acid Derivatives with Immunosuppressive Activity from the Coral-Derived Fungus Penicillium bialowiezense
    Article Snippet: .. After the recombinant plasmids were verified by sequencing, the plasmid was transformed into Escherichia coli BL21(DE3) (Invitrogen), which was grown in LB medium at 37 °C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) and grown at 20 °C for 16 h. The cell pellet was harvested and re-suspended in 30 mL buffer (20 mM Tris pH 8.5, 200 mM NaCl, and 10 mM imidazole), followed by disruption on a French press. .. Cell debris was removed by centrifugation at 21,000 rpm for 30 min.

    Article Title: Prime-boost and recombinant protein vaccination strategies using Sm-p80 protects against Schistosoma mansoni infection in the mouse model to levels previously attainable only by the irradiated cercarial vaccine
    Article Snippet: Full-length coding sequence of Sm-p80 ( ) was cloned into pCold II vector (GenScript Corp., Piscataway, NJ, USA). .. Escherichia coli BL21 (DE3; Invitrogen Corp., Carlsbad, CA, USA) were used as the expression host strain.

    Article Title: Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet
    Article Snippet: Paragraph title: Identification of GG1 based on amino acid sequence data, cloning, and expression of rGG1 ... The pET-GG1 construct was transformed into E coli BL21 (DE3; Invitrogen, Carlsbad, Calif), and rGG1 was expressed in liquid cultures and purified, as previously described.

    Affinity Chromatography:

    Article Title: S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 [OPEN]
    Article Snippet: The pET28a-S5H construct was introduced into Escherichia coli BL21 (DE3, pLys3; Invitrogen). .. Bacterial cells containing pET28a-S5H were grown in Luria-Bertani medium containing 50 mg L−1 kanamycin at 37°C to an optical density of approximately 0.6 at 600 nm, induced with 0.5 m m isopropyl β- d -1-thiogalactoside, and then incubated at 18°C for 24 h. The recombinant S5H protein was purified by Ni-NTA affinity chromatography using a previously described method ( ).

    Fast Protein Liquid Chromatography:

    Article Title: Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme
    Article Snippet: Gene expression and protein production and purification For enzyme activity assays, pBS12067 was transformed into E. coli BL21(DE3) (Life Technologies) and grown in 1 L of lysogeny broth (LB) at 37 °C with shaking at 250 rpm until an OD600 of 0.6 was reached. .. The supernatant containing PtmA2 was purified by nickel-affinity chromatography using an ÄKTA FPLC system (GE Healthcare Biosciences) equipped with a HisTrap column.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on polymer chip
    Article Snippet: Proteins were expressed in E. coli BL21 (Invitrogen) upon induction with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.6–0.8 for 3 h at 37°C. .. Concentration and purity were assessed by Coomassi-stained polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS–PAGE) and Bradford assay using bovine serum albumen (BSA) standards.

    SDS Page:

    Article Title: Prime-boost and recombinant protein vaccination strategies using Sm-p80 protects against Schistosoma mansoni infection in the mouse model to levels previously attainable only by the irradiated cercarial vaccine
    Article Snippet: Escherichia coli BL21 (DE3; Invitrogen Corp., Carlsbad, CA, USA) were used as the expression host strain. .. The fractions were analyzed by SDS-PAGE, the fractions containing a clear single protein band were pooled, and the protein concentrations were determined.

    Article Title: Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on polymer chip
    Article Snippet: Proteins were expressed in E. coli BL21 (Invitrogen) upon induction with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.6–0.8 for 3 h at 37°C. .. Concentration and purity were assessed by Coomassi-stained polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS–PAGE) and Bradford assay using bovine serum albumen (BSA) standards.

    Plasmid Preparation:

    Article Title: LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain
    Article Snippet: .. The cloned plasmid was transformed into E. coli BL21(DE3) (Invitrogen, Carlsbad, CA, USA). ..

    Article Title: Mycophenolic Acid Derivatives with Immunosuppressive Activity from the Coral-Derived Fungus Penicillium bialowiezense
    Article Snippet: .. After the recombinant plasmids were verified by sequencing, the plasmid was transformed into Escherichia coli BL21(DE3) (Invitrogen), which was grown in LB medium at 37 °C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) and grown at 20 °C for 16 h. The cell pellet was harvested and re-suspended in 30 mL buffer (20 mM Tris pH 8.5, 200 mM NaCl, and 10 mM imidazole), followed by disruption on a French press. .. Cell debris was removed by centrifugation at 21,000 rpm for 30 min.

    Article Title: Prime-boost and recombinant protein vaccination strategies using Sm-p80 protects against Schistosoma mansoni infection in the mouse model to levels previously attainable only by the irradiated cercarial vaccine
    Article Snippet: Full-length coding sequence of Sm-p80 ( ) was cloned into pCold II vector (GenScript Corp., Piscataway, NJ, USA). .. Escherichia coli BL21 (DE3; Invitrogen Corp., Carlsbad, CA, USA) were used as the expression host strain.

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: .. It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells. .. The induction of recombinant CaEno1 protein (rCaEno1) was achieved with 0.5 mm isopropyl β-d -thiogalactopyranoside for 24 h at 23 °C in Luria-Bertani medium at 200 rpm.

    Article Title: Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on polymer chip
    Article Snippet: The pMAL vector was used for bacterial expression of the proteins as fusions with an N-terminal maltose binding protein (MBP) as a purification tag. .. Proteins were expressed in E. coli BL21 (Invitrogen) upon induction with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.6–0.8 for 3 h at 37°C.

    Article Title: Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y
    Article Snippet: Expression and purification of rMHJ_0659 and rMHJ_0522 The mhj_0659 and mhj_0522 genes encoding PepP and PepF respectively were ligated into expression vector PS100030, conveying both a hexahistidine tag and ampicillin resistance, by Blue Heron Biotech (USA). .. The recombinant construct was transformed into E. coli BL21 (Invitrogen) as per the manufacturer instructions and grown overnight in LB supplemented with 100 mg/mL ampicillin.

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: .. It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells. .. The induction of recombinant CaEno1 protein (rCaEno1) was achieved with 0.5 m m isopropyl β- d -thiogalactopyranoside for 24 h at 23 °C in Luria-Bertani medium at 200 rpm.

    Recombinant:

    Article Title: LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain
    Article Snippet: Paragraph title: Production of recombinant proteins. ... The cloned plasmid was transformed into E. coli BL21(DE3) (Invitrogen, Carlsbad, CA, USA).

    Article Title: S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 [OPEN]
    Article Snippet: The S5H/DMR6 coding sequence was PCR amplified using a pair of primers, S5H_ Bam HI (5′-TTTAAGGATCCATGGCGGCAAAGCTGATATC-3′) and S5H_ Sal I (5′-CATGGTCGACTTAGTTGTTTAGAAAATTCTCGA-3′), and cloned into pET28a (Novagen) to form pET28a-S5H and produce the His-tagged recombinant S5H/DMR6 protein. .. The pET28a-S5H construct was introduced into Escherichia coli BL21 (DE3, pLys3; Invitrogen).

    Article Title: IscR Regulation of Type 3 Fimbriae Expression in Klebsiella pneumoniae CG43
    Article Snippet: .. Purification of IscR::His6 and IscR3CA ::His6 The plasmids, pET30b-IscR and pET30b-IscR3CA , in E. coli BL21(DE3)[pLysS] (Invitrogen, United States) was used to overexpress the recombinant proteins IscR::His6 and IscR3CA ::His6 , respectively. ..

    Article Title: Mycophenolic Acid Derivatives with Immunosuppressive Activity from the Coral-Derived Fungus Penicillium bialowiezense
    Article Snippet: .. After the recombinant plasmids were verified by sequencing, the plasmid was transformed into Escherichia coli BL21(DE3) (Invitrogen), which was grown in LB medium at 37 °C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) and grown at 20 °C for 16 h. The cell pellet was harvested and re-suspended in 30 mL buffer (20 mM Tris pH 8.5, 200 mM NaCl, and 10 mM imidazole), followed by disruption on a French press. .. Cell debris was removed by centrifugation at 21,000 rpm for 30 min.

    Article Title: The Candida albicans ENO1 gene encodes a transglutaminase involved in growth, cell division, morphogenesis, and osmotic protection
    Article Snippet: Paragraph title: Expression of recombinant C. albicans Eno1 protein and generation of anti-rCaEno1 polyclonal antibodies ... It was then subcloned into the pColdII (Takara) plasmid in the mentioned restriction sites to generate the pColdII/CaEno1 plasmid, which was transformed into E. coli BL21 (DE3)pLysS (Invitrogen) competent cells.

    Article Title: Human C1q Induces Apoptosis in an Ovarian Cancer Cell Line via Tumor Necrosis Factor Pathway
    Article Snippet: Paragraph title: Recombinant Expression and Purification of ghA, ghB, and ghC Modules of Human C1q ... Expression constructs pKBM-A, pKBM-B, and pKBM-C were transformed into E. coli BL21 (Invitrogen) cells in the presence of ampicillin (100 µg/ml).

    Article Title: Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y
    Article Snippet: .. The recombinant construct was transformed into E. coli BL21 (Invitrogen) as per the manufacturer instructions and grown overnight in LB supplemented with 100 mg/mL ampicillin. .. LB supplemented with 100 mg/mL ampicillin was inoculated with overnight transformation culture and grown until mid-log growth phase.

    Concentration Assay:

    Article Title: S5H/DMR6 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 Encodes a Salicylic Acid 5-Hydroxylase That Fine-Tunes Salicylic Acid Homeostasis 1 [OPEN]
    Article Snippet: The pET28a-S5H construct was introduced into Escherichia coli BL21 (DE3, pLys3; Invitrogen). .. DTT (final concentration, 2 m m ) was added to the enzyme solution, and the protein was immediately stored at −80°C.

    Article Title: The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading
    Article Snippet: Pex1/Pex6 purification Pex1-FLAG and His-Pex6 wild type and mutant complexes were co-expressed in E. coli BL21* (ThermoFisher Scientific) from the pETDuet and pCOLADuet vectors. .. The expression strain was grown in 6 L of DYT (16 g tryptone, 10 g yeast extract, 5 g NaCl) and appropriate antibiotics at 30 °C and induced at OD600 = 0.6–0.9 with IPTG (final concentration C f =0.3 mM) before overnight incubation at 18 °C.

    Article Title: Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on polymer chip
    Article Snippet: Proteins were expressed in E. coli BL21 (Invitrogen) upon induction with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) at an OD600 of 0.6–0.8 for 3 h at 37°C. .. Concentration and purity were assessed by Coomassi-stained polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS–PAGE) and Bradford assay using bovine serum albumen (BSA) standards.

    Lysis:

    Article Title: LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain
    Article Snippet: The cloned plasmid was transformed into E. coli BL21(DE3) (Invitrogen, Carlsbad, CA, USA). .. After thawing, the cells were resuspended in a lysis buffer containing 200 mM NaCl and 50 mM Tris-Cl (pH 8.0) and disrupted by sonication (Sonifier 250; Branson, Danbury, CT, USA).

    Article Title: Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme
    Article Snippet: Gene expression and protein production and purification For enzyme activity assays, pBS12067 was transformed into E. coli BL21(DE3) (Life Technologies) and grown in 1 L of lysogeny broth (LB) at 37 °C with shaking at 250 rpm until an OD600 of 0.6 was reached. .. After harvesting the cells by centrifugation at 4000 g for 15 min at 4 °C, the pellet was resuspended in lysis buffer (100 mM Tris, pH 8.0, containing 300 mM NaCl, 15 mM imidazole, and 10% glycerol), lysed by sonication, and centrifuged at 15,000 g for 20 min at 4 °C.

    Article Title: Human C1q Induces Apoptosis in an Ovarian Cancer Cell Line via Tumor Necrosis Factor Pathway
    Article Snippet: Expression constructs pKBM-A, pKBM-B, and pKBM-C were transformed into E. coli BL21 (Invitrogen) cells in the presence of ampicillin (100 µg/ml). .. Subsequently, the cell pellet for each fusion protein was lysed in 50 ml lysis buffer (20 mM Tris–HCl, pH 8.0, 0.5 M NaCl, 0.2% v/v Tween 20, 1 mM EGTA pH 7.5, 1 mM EDTA pH 7.5, and 5% v/v glycerol) containing lysozyme (100 µg/ml, Sigma-Aldrich, UK) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, UK) at 4°C for 30 min.

    Article Title: Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y
    Article Snippet: The recombinant construct was transformed into E. coli BL21 (Invitrogen) as per the manufacturer instructions and grown overnight in LB supplemented with 100 mg/mL ampicillin. .. Pellets were resuspended in Lysis Buffer [50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8) at 2 mL per gram of wet weight and lysozyme (Sigma Aldrich) was added at 1 mg/mL to break down peptidoglycan.

    Positron Emission Tomography:

    Article Title: Mycophenolic Acid Derivatives with Immunosuppressive Activity from the Coral-Derived Fungus Penicillium bialowiezense
    Article Snippet: The gene encoding IMPDH2 was cloned into the pET-28a vector (Novagen). .. After the recombinant plasmids were verified by sequencing, the plasmid was transformed into Escherichia coli BL21(DE3) (Invitrogen), which was grown in LB medium at 37 °C to an OD600 (0.8–1.0) and induced by 0.4 mM isopropyl-D-thiogalactopyranoside (IPTG) and grown at 20 °C for 16 h. The cell pellet was harvested and re-suspended in 30 mL buffer (20 mM Tris pH 8.5, 200 mM NaCl, and 10 mM imidazole), followed by disruption on a French press.

    Article Title: Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet
    Article Snippet: .. The pET-GG1 construct was transformed into E coli BL21 (DE3; Invitrogen, Carlsbad, Calif), and rGG1 was expressed in liquid cultures and purified, as previously described. .. Multiple sequence alignment of GG1 with homologous proteins from other cereals An NCBI Protein blast ( http://blast.ncbi.nlm.nih.gov ) search of GG1 (SwissProt ID: P08453.1) against spelt (Triticum spelta) , rye (Secale cereale) , barley (Hordeum vulgare L) , maize (Zea mays) , rice (Oryza sativa) , and oat (Avena sativa) was performed.

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