Structured Review

GE Healthcare coli bl21
Construction and detection of the CFA/I/II/IV-STa -toxoid -dmLT MEFA. (A) Construction of the CFA/I/II/IV MEFA. The most antigenic epitopes of the CS1, CS2, CS3, CS4, CS5 and CS6 major structural subunits were embedded into CFA/I major subunit by replacing the CfaB surface-exposed but less antigenic epitopes. (B) Construction of the 3xSTa N12S -dmLT toxoid fusion. Three copies of the STa toxoid STa N12S gene were genetically fused to the monomeric dmLT (LT R192G/L211A ) gene using SOE (splicing overlap extension) PCRs. (C) Construction of CFA/I/II/IV-STa N12S -dmLT MEFA. A substitution of the first 150 amino acids of the 3xSTa N12S -dmLT (the N-terminal STa N12S and the first 131 amino acids of LT-A subunit) with the CFA/I/II/IV MEFA created the CFA/I/II/IV-STa N12S -dmLT MEFA. Four linkers: LGA, GPVD, Gly-Pro linker GPGP, and L-linker were used for the construction. (D) Western blot to detect the CFA/I/II/IV-STa N12S -dmLT MEFA protein with anti-CFA/I, anti-CS1, -CS2, -CS3, -CS4, -CS5, and anti-CS6 MAb hybridoma supernatant (1:100; provided by Dr. AM Svennerholm), and rabbit anti-CT (1:3300; Sigma) and anti-STa antiserum (1:3300; provided by Dr. DC Robertson). Extracted MEFA proteins separated in 12% PAGE gel were detected with each anti-adhesin MAb, anti-CT and anti-STa antiserum and IRDye-labeled goat anti-mouse IgG or anti-rabbit IgG (1:5000; LI-COR). Lane (+) indicated the CFA/I/II/IV-STa N12S -dmLT MEFA proteins, whereas lane (-) of extracted total proteins of E . coli <t>BL21</t> host strain as the negative control. Lane M is the protein marker (in kilo Daltons; Precision Plus Protein pre-stained standards; Bio-Rad).
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Images

1) Product Images from "Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity"

Article Title: Genetic Fusions of a CFA/I/II/IV MEFA (Multiepitope Fusion Antigen) and a Toxoid Fusion of Heat-Stable Toxin (STa) and Heat-Labile Toxin (LT) of Enterotoxigenic Escherichia coli (ETEC) Retain Broad Anti-CFA and Antitoxin Antigenicity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0121623

Construction and detection of the CFA/I/II/IV-STa -toxoid -dmLT MEFA. (A) Construction of the CFA/I/II/IV MEFA. The most antigenic epitopes of the CS1, CS2, CS3, CS4, CS5 and CS6 major structural subunits were embedded into CFA/I major subunit by replacing the CfaB surface-exposed but less antigenic epitopes. (B) Construction of the 3xSTa N12S -dmLT toxoid fusion. Three copies of the STa toxoid STa N12S gene were genetically fused to the monomeric dmLT (LT R192G/L211A ) gene using SOE (splicing overlap extension) PCRs. (C) Construction of CFA/I/II/IV-STa N12S -dmLT MEFA. A substitution of the first 150 amino acids of the 3xSTa N12S -dmLT (the N-terminal STa N12S and the first 131 amino acids of LT-A subunit) with the CFA/I/II/IV MEFA created the CFA/I/II/IV-STa N12S -dmLT MEFA. Four linkers: LGA, GPVD, Gly-Pro linker GPGP, and L-linker were used for the construction. (D) Western blot to detect the CFA/I/II/IV-STa N12S -dmLT MEFA protein with anti-CFA/I, anti-CS1, -CS2, -CS3, -CS4, -CS5, and anti-CS6 MAb hybridoma supernatant (1:100; provided by Dr. AM Svennerholm), and rabbit anti-CT (1:3300; Sigma) and anti-STa antiserum (1:3300; provided by Dr. DC Robertson). Extracted MEFA proteins separated in 12% PAGE gel were detected with each anti-adhesin MAb, anti-CT and anti-STa antiserum and IRDye-labeled goat anti-mouse IgG or anti-rabbit IgG (1:5000; LI-COR). Lane (+) indicated the CFA/I/II/IV-STa N12S -dmLT MEFA proteins, whereas lane (-) of extracted total proteins of E . coli BL21 host strain as the negative control. Lane M is the protein marker (in kilo Daltons; Precision Plus Protein pre-stained standards; Bio-Rad).
Figure Legend Snippet: Construction and detection of the CFA/I/II/IV-STa -toxoid -dmLT MEFA. (A) Construction of the CFA/I/II/IV MEFA. The most antigenic epitopes of the CS1, CS2, CS3, CS4, CS5 and CS6 major structural subunits were embedded into CFA/I major subunit by replacing the CfaB surface-exposed but less antigenic epitopes. (B) Construction of the 3xSTa N12S -dmLT toxoid fusion. Three copies of the STa toxoid STa N12S gene were genetically fused to the monomeric dmLT (LT R192G/L211A ) gene using SOE (splicing overlap extension) PCRs. (C) Construction of CFA/I/II/IV-STa N12S -dmLT MEFA. A substitution of the first 150 amino acids of the 3xSTa N12S -dmLT (the N-terminal STa N12S and the first 131 amino acids of LT-A subunit) with the CFA/I/II/IV MEFA created the CFA/I/II/IV-STa N12S -dmLT MEFA. Four linkers: LGA, GPVD, Gly-Pro linker GPGP, and L-linker were used for the construction. (D) Western blot to detect the CFA/I/II/IV-STa N12S -dmLT MEFA protein with anti-CFA/I, anti-CS1, -CS2, -CS3, -CS4, -CS5, and anti-CS6 MAb hybridoma supernatant (1:100; provided by Dr. AM Svennerholm), and rabbit anti-CT (1:3300; Sigma) and anti-STa antiserum (1:3300; provided by Dr. DC Robertson). Extracted MEFA proteins separated in 12% PAGE gel were detected with each anti-adhesin MAb, anti-CT and anti-STa antiserum and IRDye-labeled goat anti-mouse IgG or anti-rabbit IgG (1:5000; LI-COR). Lane (+) indicated the CFA/I/II/IV-STa N12S -dmLT MEFA proteins, whereas lane (-) of extracted total proteins of E . coli BL21 host strain as the negative control. Lane M is the protein marker (in kilo Daltons; Precision Plus Protein pre-stained standards; Bio-Rad).

Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis, Labeling, Negative Control, Marker, Staining

2) Product Images from "AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs"

Article Title: AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2019.01146

Expression patterns and subcellular localization of AtPR5K2. (A) The expression of AtPR5K2 in various tissues of Arabidopsis thaliana . Total RNA was extracted from the roots, rosette leaves, cauline leaves, stems, flowers, and siliques of the wild-type plants. The transcript levels of AtPR5K2 were measured using quantitative reverse transcription PCR (qRT-PCR) and calculated relative to the expression of the endogenous control gene, TUBULIN2 . Error bars represent the ± SD from three independent experiments. (B) Subcellular localization of AtPR5K2 in Arabidopsis protoplasts. 35S:AtPR5K2-GFP and Aquaporin-RFP were coexpressed in Arabidopsis protoplasts, which were analyzed using confocal fluorescence microscopy and photographed after 24 h of incubation at 22°C. Aquaporin-RFP is a plasma-membrane marker. Scale bars represent 10 μm. (C) Subcellular localization of AtPR5K2 in the epidermal cells of tobacco ( Nicotiana benthamiana ) leave expressing 35S:AtPR5K2-GFP before and after plasmolysis. The epidermal cells were analyzed using confocal fluorescence microscopy and photographed after 48 h of incubation at 25°C. Scale bars represent 20 μm. Red asterisk indicates that AtPR5K2-GFP signal remains in the Hechtian strands. Red arrowheads point to the retracted plasma membrane. (D) In vitro kinase assays of AtPR5K2. The upper panel indicates the schematic structure of the GST-fused AtPR5K2 kinase domain (wPR5K2KD) and the GST-fused mutagenized AtPR5K2 kinase domain (mPR5K2KD). Each kinase domain was individually expressed in Escherichia coli , and 2 μg purified proteins was incubated in kinase assay buffer. Radioactive-labeled products were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and detected using radioactivity (bottom right). After electrophoresis, the purified products were stained with Coomassie brilliant blue (bottom left).
Figure Legend Snippet: Expression patterns and subcellular localization of AtPR5K2. (A) The expression of AtPR5K2 in various tissues of Arabidopsis thaliana . Total RNA was extracted from the roots, rosette leaves, cauline leaves, stems, flowers, and siliques of the wild-type plants. The transcript levels of AtPR5K2 were measured using quantitative reverse transcription PCR (qRT-PCR) and calculated relative to the expression of the endogenous control gene, TUBULIN2 . Error bars represent the ± SD from three independent experiments. (B) Subcellular localization of AtPR5K2 in Arabidopsis protoplasts. 35S:AtPR5K2-GFP and Aquaporin-RFP were coexpressed in Arabidopsis protoplasts, which were analyzed using confocal fluorescence microscopy and photographed after 24 h of incubation at 22°C. Aquaporin-RFP is a plasma-membrane marker. Scale bars represent 10 μm. (C) Subcellular localization of AtPR5K2 in the epidermal cells of tobacco ( Nicotiana benthamiana ) leave expressing 35S:AtPR5K2-GFP before and after plasmolysis. The epidermal cells were analyzed using confocal fluorescence microscopy and photographed after 48 h of incubation at 25°C. Scale bars represent 20 μm. Red asterisk indicates that AtPR5K2-GFP signal remains in the Hechtian strands. Red arrowheads point to the retracted plasma membrane. (D) In vitro kinase assays of AtPR5K2. The upper panel indicates the schematic structure of the GST-fused AtPR5K2 kinase domain (wPR5K2KD) and the GST-fused mutagenized AtPR5K2 kinase domain (mPR5K2KD). Each kinase domain was individually expressed in Escherichia coli , and 2 μg purified proteins was incubated in kinase assay buffer. Radioactive-labeled products were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and detected using radioactivity (bottom right). After electrophoresis, the purified products were stained with Coomassie brilliant blue (bottom left).

Techniques Used: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence, Microscopy, Incubation, Marker, In Vitro, Purification, Kinase Assay, Labeling, Polyacrylamide Gel Electrophoresis, SDS Page, Radioactivity, Electrophoresis, Staining

3) Product Images from "PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence"

Article Title: PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1010446

PIAS1 interacts with multiple proteins in the RNP complex of IAV. (A, B) Interaction of V5-WSNNP and Myc-PIAS1 in HEK293T cells by using a co-IP assay. HEK293T cells were individually transfected or co-transfected with plasmids expressing V5-WSNNP and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Myc mAb (B), and subjected to western blotting with a rabbit anti-V5 pAb and a rabbit anti-Myc pAb for the detection of WSNNP and PIAS1, respectively. (C) Interaction of GST-WSNNP and His-PIAS1 by using a GST pull-down assay. His-tagged PIAS1 was expressed in E . coli BL21 (DE3) and purified by using Ni Sepharose Excel resin, and the GST or GST-NP protein was expressed in HEK293T cells and purified by using Glutathione Sepharose 4 Fast Flow. An equal amount of purified PIAS1 was mixed with the Glutathione Sepharose 4 Fast Flow samples that bind GST or GST-NP. After rocking and washing, the mixed samples were separated by SDS-PAGE and stained with Coomassie blue. (D) Interaction of IAV NP and PIAS1 in virus-infected cells. HEK293T cells were transfected for 24 h to express Myc-PIAS1, and were then infected with WSN (H1N1) virus (MOI = 5). At 30 h p.i., cell lysates were immunoprecipitated with a mouse anti-NP mAb, followed by western blotting with a rabbit anti-NP pAb and a rabbit anti-Myc pAb. (E) Co-localization of IAV NP and PIAS1 in A549 cells infected with WSN (H1N1) virus. A549 cells were infected with WSN (H1N1) virus (MOI = 5). At 2, 4, 6, and 8 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb and a rabbit anti-PIAS1 pAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H+L) (red) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green). The nuclei were stained with DAPI. (F-H) Co-IP assay to examine the interactions between Myc-PIAS1 and PB2, PB1, and PA of WSN (H1N1) virus in HEK293T cells. HEK293T cells were individually transfected or co-transfected with plasmids expressing WSNPB2, WSNPB1, WSNPA, and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-Myc mAb and were subjected to western blotting with a rabbit anti-PB2 pAb (F), a rabbit anti-PB1 pAb (G), a rabbit anti-PA pAb (H), and a rabbit anti-Myc pAb (F-H) for the detection of PB2, PB1, PA, and PIAS1, respectively.
Figure Legend Snippet: PIAS1 interacts with multiple proteins in the RNP complex of IAV. (A, B) Interaction of V5-WSNNP and Myc-PIAS1 in HEK293T cells by using a co-IP assay. HEK293T cells were individually transfected or co-transfected with plasmids expressing V5-WSNNP and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Myc mAb (B), and subjected to western blotting with a rabbit anti-V5 pAb and a rabbit anti-Myc pAb for the detection of WSNNP and PIAS1, respectively. (C) Interaction of GST-WSNNP and His-PIAS1 by using a GST pull-down assay. His-tagged PIAS1 was expressed in E . coli BL21 (DE3) and purified by using Ni Sepharose Excel resin, and the GST or GST-NP protein was expressed in HEK293T cells and purified by using Glutathione Sepharose 4 Fast Flow. An equal amount of purified PIAS1 was mixed with the Glutathione Sepharose 4 Fast Flow samples that bind GST or GST-NP. After rocking and washing, the mixed samples were separated by SDS-PAGE and stained with Coomassie blue. (D) Interaction of IAV NP and PIAS1 in virus-infected cells. HEK293T cells were transfected for 24 h to express Myc-PIAS1, and were then infected with WSN (H1N1) virus (MOI = 5). At 30 h p.i., cell lysates were immunoprecipitated with a mouse anti-NP mAb, followed by western blotting with a rabbit anti-NP pAb and a rabbit anti-Myc pAb. (E) Co-localization of IAV NP and PIAS1 in A549 cells infected with WSN (H1N1) virus. A549 cells were infected with WSN (H1N1) virus (MOI = 5). At 2, 4, 6, and 8 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb and a rabbit anti-PIAS1 pAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H+L) (red) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (green). The nuclei were stained with DAPI. (F-H) Co-IP assay to examine the interactions between Myc-PIAS1 and PB2, PB1, and PA of WSN (H1N1) virus in HEK293T cells. HEK293T cells were individually transfected or co-transfected with plasmids expressing WSNPB2, WSNPB1, WSNPA, and Myc-PIAS1. Cell lysates were immunoprecipitated with a mouse anti-Myc mAb and were subjected to western blotting with a rabbit anti-PB2 pAb (F), a rabbit anti-PB1 pAb (G), a rabbit anti-PA pAb (H), and a rabbit anti-Myc pAb (F-H) for the detection of PB2, PB1, PA, and PIAS1, respectively.

Techniques Used: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Purification, SDS Page, Staining, Infection, Incubation

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    GE Healthcare escherichia coli bl21 de3 cells
    Interaction between the CSFV E2 protein and MEK2. (A) Yeast cotransformation assay. The Y2HGold yeast strain was cotransformed with pGBKT7-E2 (BD-E2)/pGADT7-MEK2 (AD-MEK2), pGBKT7-p53 (BD-p53)/pGADT7-T (AD-T) (positive control), or pGBKT7-Lamin (BD-Lamin)/AD-T (negative control). (B) Coimmunoprecipitation (Co-IP) analysis of MEK2 and E2. HEK293T <t>cells</t> were cotransfected with pMyc-MEK2 and pCAGGS-E2-Flag. Cells collected at 48 h posttransfection (hpt) were lysed, precleared with protein G-agarose, and incubated with anti-Flag M2 affinity gel for 6 h at 4°C. The proteins were analyzed by Western blotting using a rabbit anti-Flag or anti-Myc polyclonal antibody (1:500). (C) GST pulldown assay. GST or GST-E2 expressed in <t>Escherichia</t> <t>coli</t> <t>BL21(DE3)</t> was purified with a glutathione-Sepharose 4B resin (catalog no. 10049253; GE Healthcare) and incubated with Myc-MEK2 expressed in HEK293T cells. The bound proteins were subjected to Western blotting using the indicated antibodies. (D and E) Colocalization of MEK2 with E2. HEK293T cells cotransfected with pMyc-MEK2 and pCAGGS-E2-Flag (D) or PK-15 cells infected with CSFV (E) were examined by indirect immunofluorescence assay (IFA) for expression of MEK2 (red) and E2 (green).
    Escherichia Coli Bl21 De3 Cells, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 98/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Interaction between the CSFV E2 protein and MEK2. (A) Yeast cotransformation assay. The Y2HGold yeast strain was cotransformed with pGBKT7-E2 (BD-E2)/pGADT7-MEK2 (AD-MEK2), pGBKT7-p53 (BD-p53)/pGADT7-T (AD-T) (positive control), or pGBKT7-Lamin (BD-Lamin)/AD-T (negative control). (B) Coimmunoprecipitation (Co-IP) analysis of MEK2 and E2. HEK293T cells were cotransfected with pMyc-MEK2 and pCAGGS-E2-Flag. Cells collected at 48 h posttransfection (hpt) were lysed, precleared with protein G-agarose, and incubated with anti-Flag M2 affinity gel for 6 h at 4°C. The proteins were analyzed by Western blotting using a rabbit anti-Flag or anti-Myc polyclonal antibody (1:500). (C) GST pulldown assay. GST or GST-E2 expressed in Escherichia coli BL21(DE3) was purified with a glutathione-Sepharose 4B resin (catalog no. 10049253; GE Healthcare) and incubated with Myc-MEK2 expressed in HEK293T cells. The bound proteins were subjected to Western blotting using the indicated antibodies. (D and E) Colocalization of MEK2 with E2. HEK293T cells cotransfected with pMyc-MEK2 and pCAGGS-E2-Flag (D) or PK-15 cells infected with CSFV (E) were examined by indirect immunofluorescence assay (IFA) for expression of MEK2 (red) and E2 (green).

    Journal: Journal of Virology

    Article Title: Mitogen-Activated Protein Kinase Kinase 2, a Novel E2-Interacting Protein, Promotes the Growth of Classical Swine Fever Virus via Attenuation of the JAK-STAT Signaling Pathway

    doi: 10.1128/JVI.01407-16

    Figure Lengend Snippet: Interaction between the CSFV E2 protein and MEK2. (A) Yeast cotransformation assay. The Y2HGold yeast strain was cotransformed with pGBKT7-E2 (BD-E2)/pGADT7-MEK2 (AD-MEK2), pGBKT7-p53 (BD-p53)/pGADT7-T (AD-T) (positive control), or pGBKT7-Lamin (BD-Lamin)/AD-T (negative control). (B) Coimmunoprecipitation (Co-IP) analysis of MEK2 and E2. HEK293T cells were cotransfected with pMyc-MEK2 and pCAGGS-E2-Flag. Cells collected at 48 h posttransfection (hpt) were lysed, precleared with protein G-agarose, and incubated with anti-Flag M2 affinity gel for 6 h at 4°C. The proteins were analyzed by Western blotting using a rabbit anti-Flag or anti-Myc polyclonal antibody (1:500). (C) GST pulldown assay. GST or GST-E2 expressed in Escherichia coli BL21(DE3) was purified with a glutathione-Sepharose 4B resin (catalog no. 10049253; GE Healthcare) and incubated with Myc-MEK2 expressed in HEK293T cells. The bound proteins were subjected to Western blotting using the indicated antibodies. (D and E) Colocalization of MEK2 with E2. HEK293T cells cotransfected with pMyc-MEK2 and pCAGGS-E2-Flag (D) or PK-15 cells infected with CSFV (E) were examined by indirect immunofluorescence assay (IFA) for expression of MEK2 (red) and E2 (green).

    Article Snippet: GST-tagged MEK2 mutants were expressed in Escherichia coli BL21(DE3) cells and incubated with glutathione-Sepharose 4B resin (catalog no. 17-0756-01; GE Healthcare).

    Techniques: Positive Control, Negative Control, Co-Immunoprecipitation Assay, Incubation, Western Blot, GST Pulldown Assay, Purification, Infection, Immunofluorescence, Expressing

    Construction, purification, and verification of cholera toxin B subunit (CTB)-HUUC. (A) Composition of the CTB-HUUC construct. (B) Visualization of the purified CTB and CTB-HUUC peptides. CTB and CTB-HUUC peptides purified from Escherichia coli BL21(DE3) transformed with pET28a(+)/ ctB-huuc , pET28a(+)/ ctB were resolved in 12% SDS-PAGE gel and stained with Coomassie Blue. (C) Identity verification of CTB and CTB-HUUC using Western blotting. CTB and CTB-HUUC peptides resolved in 12% SDS-PAGE gels were probed with rabbit anti- Helicobacter pylori polyclonal antibody, rabbit anti-urease B subunit monoclonal antibody, or mouse anti-CTB monoclonal antibody. (D) The adjuvanticity of CTB-HUUC peptide was evaluated using GM1-ELISA. *** p

    Journal: Frontiers in Immunology

    Article Title: Protection Against Helicobacter pylori Infection in BALB/c Mouse Model by Oral Administration of Multivalent Epitope-Based Vaccine of Cholera Toxin B Subunit-HUUC

    doi: 10.3389/fimmu.2018.01003

    Figure Lengend Snippet: Construction, purification, and verification of cholera toxin B subunit (CTB)-HUUC. (A) Composition of the CTB-HUUC construct. (B) Visualization of the purified CTB and CTB-HUUC peptides. CTB and CTB-HUUC peptides purified from Escherichia coli BL21(DE3) transformed with pET28a(+)/ ctB-huuc , pET28a(+)/ ctB were resolved in 12% SDS-PAGE gel and stained with Coomassie Blue. (C) Identity verification of CTB and CTB-HUUC using Western blotting. CTB and CTB-HUUC peptides resolved in 12% SDS-PAGE gels were probed with rabbit anti- Helicobacter pylori polyclonal antibody, rabbit anti-urease B subunit monoclonal antibody, or mouse anti-CTB monoclonal antibody. (D) The adjuvanticity of CTB-HUUC peptide was evaluated using GM1-ELISA. *** p

    Article Snippet: All vaccine proteins CTB-HUUC, CTB, HpaA, UreA, UreB, and CagA were purified by Ni2+ -charged column chromatography and gel filtration chromatography (GE Healthcare, USA) from Escherichia coli BL21(DE3) transformed with the respective recombinant vectors.

    Techniques: Purification, CtB Assay, Construct, Transformation Assay, SDS Page, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

    Production of GST-ROP2 fusion protein. (A) Design of fragmentation of cDNA of ROP2. Eight fragments of ROP2 were cloned as

    Journal: The Korean Journal of Parasitology

    Article Title: High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles

    doi: 10.3347/kjp.2014.52.4.367

    Figure Lengend Snippet: Production of GST-ROP2 fusion protein. (A) Design of fragmentation of cDNA of ROP2. Eight fragments of ROP2 were cloned as "A", full sequence of ROP2 without signal sequence; "B", 1/2 Nt sequence of ROP2; "C", 1/2 Ct containing kinase domain; "D", 1/4 Nt; "E", middle 1/4 Ct; "G", 1/4Ct; "H", 1/2 Nt of kinase domain; and "I", 1/2 Ct of kinase domain. (B) Expression of recombinant ROP2 antigens. All recombinant proteins were well induced and tested by western blot with anti-GST antibody. (C) Antigenicity of recombinant ROP2 antigens. The antigenicity was tested by western blot against patient serum. (D) Solubility of recombinant ROP2 antigens. Solubility of rGST-ROP2 324-561 induced was confirmed by western blot with anti-GST antibody. (E) Expression of recombinant linker ROP2 antigens. "L", lysate of BL21 (DE3) pLysS E. coli transformed with pGEX-4T-1/GRA2 31-71 ; "R2", that with pGEX-4T-1/ROP2 324-561 ; and "LR", that with pGEX-4T-1/GRA2 31-71 -ROP2 324-561 . The expression was confirmed by western blot with anti-GST antibody. (F) Antigenicity of recombinant linker ROP2 antigens. It was tested by western blot against patient serum. (G) Solubility of recombinant linker ROP2 antigens. Soluble and insoluble fractions of rGST-GRA2 31-71 -ROP2 324-561 protein were tested by western blot.

    Article Snippet: The pGEX-4T-1 and recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli .

    Techniques: Clone Assay, Sequencing, Expressing, Recombinant, Western Blot, Solubility, Transformation Assay

    Production of GST-GRA2 fusion protein.

    Journal: The Korean Journal of Parasitology

    Article Title: High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles

    doi: 10.3347/kjp.2014.52.4.367

    Figure Lengend Snippet: Production of GST-GRA2 fusion protein. "N" indicates lysate of BL21 (DE3) pLysS E. coli without induction; "G", lysate of E. coli transformed with vector after induction; "R", Toxoplasma lysate antigen (TLA) of RH strain; "T", total lysates of E. coli ; "S", soluble fraction; and "P", insoluble fraction. The meaning of abbreviations is the same in the following context without extra illustration. (A) Design of fragmentation of cDNA of GRA2; 7 fragments of GRA2 were cloned. The name of clones and amino acid regions are indicated. "F", full sequence of GRA2 without signal sequence; "A", 2/3 N-terminal (Nt); "B", 2/3 C-terminal (Ct); "C", half Nt; "D", half Ct; "E", middle 1/3 sequence; and "L", linker, is the high disorder sequence of Nt. (B) Expression of recombinant GRA2 antigens induced at 30℃, 0.5 mM IPTG. Target bands against GST by western blot were marked with asterisks. (C) Antigenicity of recombinant GRA2 antigens. Patient serum was applied to detect the antigenicity of recombinant proteins against human IgG by western blot. The detectable signal was marked with asterisks. (D) Solubility of recombinant GRA2 antigens. The solubility of rGST-GRA2 25-105 was tested by western blot against GST.

    Article Snippet: The pGEX-4T-1 and recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli .

    Techniques: Transformation Assay, Plasmid Preparation, Clone Assay, Sequencing, Expressing, Recombinant, Western Blot, Solubility

    Production of GST-MIC2 fusion protein. (A) Design of fragmentation of cDNA of MIC2. Ten fragments of MIC2 were cloned as

    Journal: The Korean Journal of Parasitology

    Article Title: High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles

    doi: 10.3347/kjp.2014.52.4.367

    Figure Lengend Snippet: Production of GST-MIC2 fusion protein. (A) Design of fragmentation of cDNA of MIC2. Ten fragments of MIC2 were cloned as "F", full sequence of MIC2; "A", full sequence without Ct transmembrane region; "B", 2/3 Nt; "C", 2/3 Ct; "D", 1/3 Nt; "E", middle 1/3; "G", 1/3 Ct; "H", half Nt of D; "I", half Ct of D; and "J", middle 1/3 of D. (B) Expression of recombinant MIC2 antigens. All recombinant proteins were well induced and tested by western blot against anti-GST antibody. (C) Antigenicity of recombinant MIC2 antigens. The antigenicity of GST-MIC2 was tested by western blot against patient serum. (D) Solubility of recombinant MIC2 antigens. Total lysate, soluble, and insoluble fractions of rGST-MIC2 1-284 were detected against GST antibody. (E) Expression of recombinant linker MIC2 antigens. BL21 (DE3) pLysS E. coli containing GST and GST linker MIC2 1-284 recombinant plasmids were induced; "L", lysate transformed with pGEX-4T-1/GRA2 31-71 ; "M2", that with pGEX-4T-1/MIC2 1-284 ; and "LM", that with pGEX-4T-1/GRA2 31-71 -MIC2 1-284 . The expression was confirmed against anti-GST antibody. (F) Antigenicity of recombinant linker MIC2 antigens. It was tested against patient serum. (G) Solubility of recombinant linker MIC2 antigens. The soluble and insoluble fractions of rGST-GRA2 31-71 -MIC2 1-284 protein were tested by western blot.

    Article Snippet: The pGEX-4T-1 and recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli .

    Techniques: Clone Assay, Sequencing, Expressing, Recombinant, Western Blot, Solubility, Transformation Assay

    Construction and detection of LT R192G -STb-Stx2e-STa P12F toxoid MEFA. (A) Schematic illustration of the toxoid MEFA gene. Nucleotides coding STb epitope (KKDLCEHY), Stx2e A subunit epitope (QSYVSSLN), and full-length STa toxoid STa P12F substituted nucleotides coding the 52–59, 77–83, and 138–154 amino acids of the A1 peptide of the monomeric LT toxoid LT R192G to generate a toxoid MEFA gene using splicing overlap extension (SOE) PCRs. PCR primers: 1, T7-F; 2, STb-LT A Chim-R; 3, LT A -STbChim-F; 4, Stx2e-LT A -R; 5, LT A -Stx2e-F; 6, LT 192 -STa P12F -R; 7, LT 192 -STa P12F -F; 8, T7-R. Two PCRs using primers LT 192 NheI-F3 and 6, primers 7 and LT 192 BamH1-R1 constructed the LT R192G -STa P12F chimeric gene initially. The second set of two PCRs using primers 1 and 2, primers 3 and 8 to insert the STb epitope into the LT R192G -STa P12F toxoid chimeric gene, and the third set of two PCRs with primers 1 and 4, primers 5 and 8 to insert the Ste2e A subunit epitope into the LT R192G -STb-STa P12F chimeric gene. (B) Western blots to show detection of the toxoid MEFA protein with rabbit anti-CT (1:3000; Sigma), anti-STa (1:5000), anti-STb (1:1000), and anti-Stx2e (1:3000) antiserum, with IRDye-labeled goat-anti-rabbit IgG (1:5000; LI-COR) as the secondary antibody. Lane M is the protein marker (in kilo Daltons; Precision Plus Protein Pre-stained standard; Bio-Rad), 9161 represented proteins extracted from the toxoid MEFA recombinant strain, and (−) indicated proteins extracted from E. coli BL21 host strain.

    Journal: Veterinary Microbiology

    Article Title: Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC)

    doi: 10.1016/j.vetmic.2016.02.002

    Figure Lengend Snippet: Construction and detection of LT R192G -STb-Stx2e-STa P12F toxoid MEFA. (A) Schematic illustration of the toxoid MEFA gene. Nucleotides coding STb epitope (KKDLCEHY), Stx2e A subunit epitope (QSYVSSLN), and full-length STa toxoid STa P12F substituted nucleotides coding the 52–59, 77–83, and 138–154 amino acids of the A1 peptide of the monomeric LT toxoid LT R192G to generate a toxoid MEFA gene using splicing overlap extension (SOE) PCRs. PCR primers: 1, T7-F; 2, STb-LT A Chim-R; 3, LT A -STbChim-F; 4, Stx2e-LT A -R; 5, LT A -Stx2e-F; 6, LT 192 -STa P12F -R; 7, LT 192 -STa P12F -F; 8, T7-R. Two PCRs using primers LT 192 NheI-F3 and 6, primers 7 and LT 192 BamH1-R1 constructed the LT R192G -STa P12F chimeric gene initially. The second set of two PCRs using primers 1 and 2, primers 3 and 8 to insert the STb epitope into the LT R192G -STa P12F toxoid chimeric gene, and the third set of two PCRs with primers 1 and 4, primers 5 and 8 to insert the Ste2e A subunit epitope into the LT R192G -STb-STa P12F chimeric gene. (B) Western blots to show detection of the toxoid MEFA protein with rabbit anti-CT (1:3000; Sigma), anti-STa (1:5000), anti-STb (1:1000), and anti-Stx2e (1:3000) antiserum, with IRDye-labeled goat-anti-rabbit IgG (1:5000; LI-COR) as the secondary antibody. Lane M is the protein marker (in kilo Daltons; Precision Plus Protein Pre-stained standard; Bio-Rad), 9161 represented proteins extracted from the toxoid MEFA recombinant strain, and (−) indicated proteins extracted from E. coli BL21 host strain.

    Article Snippet: E. coli strains BL21 (GE Healthcare, Piscataway, NJ) and DH5α (Promega, Madison, WI) were used to express toxoid MEFA and MBP fusion proteins.

    Techniques: Polymerase Chain Reaction, Construct, Western Blot, Labeling, Marker, Staining, Recombinant