shuffle t7  (New England Biolabs)


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    Structured Review

    New England Biolabs shuffle t7
    Esterase activity of cytoplasmic extracts from Escherichia coli <t>SHuffle</t> T7 transformed with plasmid pET without ( E. coli SHuffle T7-pET) or with ( E. coli SHuffle T7-pET-sub1) the sub1 insert and exposed to various concentrations of IPTG. Activity is expressed as the concentration of p -nitrophenol released from p -nitrophenyl butyrate substrate in 5- and 30-min reactions. These results are the means of five replicates±SD. Bar values accompanied by the same lower case letter or upper case letter were not significantly different.
    Shuffle T7, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/shuffle t7/product/New England Biolabs
    Average 96 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    shuffle t7 - by Bioz Stars, 2022-08
    96/100 stars

    Images

    1) Product Images from "Enzymatic Degradation of p-Nitrophenyl Esters, Polyethylene Terephthalate, Cutin, and Suberin by Sub1, a Suberinase Encoded by the Plant Pathogen Streptomyces scabies"

    Article Title: Enzymatic Degradation of p-Nitrophenyl Esters, Polyethylene Terephthalate, Cutin, and Suberin by Sub1, a Suberinase Encoded by the Plant Pathogen Streptomyces scabies

    Journal: Microbes and Environments

    doi: 10.1264/jsme2.ME19086

    Esterase activity of cytoplasmic extracts from Escherichia coli SHuffle T7 transformed with plasmid pET without ( E. coli SHuffle T7-pET) or with ( E. coli SHuffle T7-pET-sub1) the sub1 insert and exposed to various concentrations of IPTG. Activity is expressed as the concentration of p -nitrophenol released from p -nitrophenyl butyrate substrate in 5- and 30-min reactions. These results are the means of five replicates±SD. Bar values accompanied by the same lower case letter or upper case letter were not significantly different.
    Figure Legend Snippet: Esterase activity of cytoplasmic extracts from Escherichia coli SHuffle T7 transformed with plasmid pET without ( E. coli SHuffle T7-pET) or with ( E. coli SHuffle T7-pET-sub1) the sub1 insert and exposed to various concentrations of IPTG. Activity is expressed as the concentration of p -nitrophenol released from p -nitrophenyl butyrate substrate in 5- and 30-min reactions. These results are the means of five replicates±SD. Bar values accompanied by the same lower case letter or upper case letter were not significantly different.

    Techniques Used: Activity Assay, Transformation Assay, Plasmid Preparation, Positron Emission Tomography, Concentration Assay

    SDS-PAGE gel of the cytoplasmic extract obtained from pET-transformed  Escherichia coli  strain SHuffle T7, without ( E. coli  SHuffle T7-pET) or with ( E. coli  SHuffle T7-pET- sub1 ) the insert of the  sub1  gene, after induction with different concentrations of IPTG.
    Figure Legend Snippet: SDS-PAGE gel of the cytoplasmic extract obtained from pET-transformed Escherichia coli strain SHuffle T7, without ( E. coli SHuffle T7-pET) or with ( E. coli SHuffle T7-pET- sub1 ) the insert of the sub1 gene, after induction with different concentrations of IPTG.

    Techniques Used: SDS Page, Positron Emission Tomography, Transformation Assay

    SDS–PAGE gel of cytoplasmic soluble proteins obtained from  Escherichia coli  transformed with SHuffle T7-pET- sub1 , after fractionation on the affinity column (IMAC). Lane 1, molecular weight marker; lane 2, cytoplasmic extract; lane 3, flow-through; lane 4, proteins released after washing with buffer A; lanes 5 to 9, proteins released after washing with buffer A supplemented with 4, 5, 10, 50, or 200‍ ‍mM imidazole, respectively.
    Figure Legend Snippet: SDS–PAGE gel of cytoplasmic soluble proteins obtained from Escherichia coli transformed with SHuffle T7-pET- sub1 , after fractionation on the affinity column (IMAC). Lane 1, molecular weight marker; lane 2, cytoplasmic extract; lane 3, flow-through; lane 4, proteins released after washing with buffer A; lanes 5 to 9, proteins released after washing with buffer A supplemented with 4, 5, 10, 50, or 200‍ ‍mM imidazole, respectively.

    Techniques Used: SDS Page, Transformation Assay, Positron Emission Tomography, Fractionation, Affinity Column, Molecular Weight, Marker

    2) Product Images from "The cysteine-rich exosporium morphogenetic protein, CdeC, exhibits self-assembly properties that lead to organized inclusion bodies in Escherichia coli"

    Article Title: The cysteine-rich exosporium morphogenetic protein, CdeC, exhibits self-assembly properties that lead to organized inclusion bodies in Escherichia coli

    Journal: bioRxiv

    doi: 10.1101/2020.07.09.196287

    Effect of recombinant strain type in the soluble expression and structural organization of CdeC. A) Recombinant CdeC expression in E. coli BL21 (DE3) pRIL and SHuffle T7 strains carrying pARR19 were induced with 0.5 mM IPTG for 16 h at 37°C. Cells were collected and lysed in soluble (S) and insoluble (I) lysis buffer, electrophoresed, and analyzed by Western blot as described in the Methods section. Each lane was loaded with 2 μg of protein lysate. Molecular mass (kDa) markers are indicated at the left side of the panels, and molecular mass of the detected His-tagged immunoreactive bands are indicated at the right side of the panels. B) Thin sections of E. coli BL21 (DE3) pRIL and E. coli SHuffle T7 expressing CdeC from C. difficile R20291 were analyzed by transmission electron microscopy as described in the Method section. Representative micrographs of several E. coli cells are shown in the upper panel. The middle panel shows selected individual cells, and the lower panel displays a magnified view of the thin section of inclusion bodies inside E. coli BL21 (DE3) pRIL or SHuffle T7. When corresponding, lamellae-like formation in CdeC is indicated with blue arrowheads.
    Figure Legend Snippet: Effect of recombinant strain type in the soluble expression and structural organization of CdeC. A) Recombinant CdeC expression in E. coli BL21 (DE3) pRIL and SHuffle T7 strains carrying pARR19 were induced with 0.5 mM IPTG for 16 h at 37°C. Cells were collected and lysed in soluble (S) and insoluble (I) lysis buffer, electrophoresed, and analyzed by Western blot as described in the Methods section. Each lane was loaded with 2 μg of protein lysate. Molecular mass (kDa) markers are indicated at the left side of the panels, and molecular mass of the detected His-tagged immunoreactive bands are indicated at the right side of the panels. B) Thin sections of E. coli BL21 (DE3) pRIL and E. coli SHuffle T7 expressing CdeC from C. difficile R20291 were analyzed by transmission electron microscopy as described in the Method section. Representative micrographs of several E. coli cells are shown in the upper panel. The middle panel shows selected individual cells, and the lower panel displays a magnified view of the thin section of inclusion bodies inside E. coli BL21 (DE3) pRIL or SHuffle T7. When corresponding, lamellae-like formation in CdeC is indicated with blue arrowheads.

    Techniques Used: Recombinant, Expressing, Lysis, Western Blot, Transmission Assay, Electron Microscopy

    3) Product Images from "Recombinant production of growth factors for application in cell culture"

    Article Title: Recombinant production of growth factors for application in cell culture

    Journal: bioRxiv

    doi: 10.1101/2022.02.15.480596

    Expression systems for recombinant GF production. (A) Small-scale protein expression screening used to identify the expression vector and host strain combination capable of facilitating cytoplasmic soluble protein expression. The band corresponding to the protein of interest is marked with (*). T - total cell lysate; S - soluble fraction. (B) Expression vector and host strain combinations for successful expression and purification of soluble, bioactive growth factors. (^) denotes instances where the use of SHuffle T7 Express was required for soluble expression of some orthologs.
    Figure Legend Snippet: Expression systems for recombinant GF production. (A) Small-scale protein expression screening used to identify the expression vector and host strain combination capable of facilitating cytoplasmic soluble protein expression. The band corresponding to the protein of interest is marked with (*). T - total cell lysate; S - soluble fraction. (B) Expression vector and host strain combinations for successful expression and purification of soluble, bioactive growth factors. (^) denotes instances where the use of SHuffle T7 Express was required for soluble expression of some orthologs.

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Purification

    Recombinant GF production. Scale up of protein expressions for (A) FGF-2 AND FGF-1 cloned in pMCSG53 vector with N-terminal His6x tag and expressed in BL21(DE3) Gold cells. Targets include F1 (FGF2_Atlantic salmon); F2 (FGF2_Pufferfish); F3 (FGF1_Sheep); F4 (FGF1_Bovine) (B) PDGF-BB expressed in SHuffle T7 express cells. Target shown is P1 (PDGFBB_Cormorant) (C) IGF-1/IGF-2 cloned in pMCSG53-His6x-DsbC /pMCSG53-His6x-SUMO and expressed in SHuffle T7 express cells. Targets include ( K1 ) IGF1_Bovine (SUMO-His6x tag); ( K2 ) IGF1_Bovine (DsbC-His6x tag); (K3 ) IGF1_Goose; (K4 ) IGF1_Frog; ( J1 ) IGF2_Human; ( J2 ) IGF2_Bovine; ( J3 ) IGF2_Nile tilapia (D) TGFβ-1 cloned in pMCSG53-His6x-DsbC and expressed in SHuffle T7 express cells. Targets shown are TGFβ1_human ( T1 ); TGFβ−1_bovine ( T2 ); TGFβ−1_chicken ( T3 ); TGFβ−1_little egret ( T4 ). UC =uncut before TEV digest; C =48h post-TEV digest; TEV protease runs at 25 kDa (marked with ^). After the TEV digest and a second Ni-NTA affinity chromatography step, the concentrated, purified FGF-2/FGF-1 runs at 15 kDa on an SDS-PAGE (marked with ) shown in (A) . PDGF-BB runs at 15 kDa corresponding to the monomer (marked with ⊇) shown in (B) . DsbC fusion IGF-1/IGF-2 runs at 35 kDa (marked with *). IGF1-SUMO runs at 20 kDa (marked with **), as seen in (C) . DsbC-TGFβ-1 runs at 40 kDa (marked with # ).
    Figure Legend Snippet: Recombinant GF production. Scale up of protein expressions for (A) FGF-2 AND FGF-1 cloned in pMCSG53 vector with N-terminal His6x tag and expressed in BL21(DE3) Gold cells. Targets include F1 (FGF2_Atlantic salmon); F2 (FGF2_Pufferfish); F3 (FGF1_Sheep); F4 (FGF1_Bovine) (B) PDGF-BB expressed in SHuffle T7 express cells. Target shown is P1 (PDGFBB_Cormorant) (C) IGF-1/IGF-2 cloned in pMCSG53-His6x-DsbC /pMCSG53-His6x-SUMO and expressed in SHuffle T7 express cells. Targets include ( K1 ) IGF1_Bovine (SUMO-His6x tag); ( K2 ) IGF1_Bovine (DsbC-His6x tag); (K3 ) IGF1_Goose; (K4 ) IGF1_Frog; ( J1 ) IGF2_Human; ( J2 ) IGF2_Bovine; ( J3 ) IGF2_Nile tilapia (D) TGFβ-1 cloned in pMCSG53-His6x-DsbC and expressed in SHuffle T7 express cells. Targets shown are TGFβ1_human ( T1 ); TGFβ−1_bovine ( T2 ); TGFβ−1_chicken ( T3 ); TGFβ−1_little egret ( T4 ). UC =uncut before TEV digest; C =48h post-TEV digest; TEV protease runs at 25 kDa (marked with ^). After the TEV digest and a second Ni-NTA affinity chromatography step, the concentrated, purified FGF-2/FGF-1 runs at 15 kDa on an SDS-PAGE (marked with ) shown in (A) . PDGF-BB runs at 15 kDa corresponding to the monomer (marked with ⊇) shown in (B) . DsbC fusion IGF-1/IGF-2 runs at 35 kDa (marked with *). IGF1-SUMO runs at 20 kDa (marked with **), as seen in (C) . DsbC-TGFβ-1 runs at 40 kDa (marked with # ).

    Techniques Used: Recombinant, Clone Assay, Plasmid Preparation, Affinity Chromatography, Purification, SDS Page

    4) Product Images from "Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli"

    Article Title: Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-014-0186-0

    SDS-PAGE and immunoblot analysis of HisDapGalNAcT2 expressed in E. coli . (A)  Origami™ 2(DE3)pLysS cells carrying plasmid pET23d(+):: HisDapGalNAcT2  were grown in LB medium at 37°C until OD 600  0.5, at which point IPTG (final concentration 1 mM) was added and cultures were incubated for a further 5 h. Cells were harvested by centrifugation, lysed and total cell lysate (T), the soluble protein fraction (S) and the insoluble particulate fraction (P) were separated by SDS-PAGE and visualised by Coomassie staining. HisDapGalNAcT2 with an estimated mass of 61.7 kDa (indicated by arrow) was not detected in the soluble (S) cell fraction, but a band of the correct size in the insoluble particulate (P) fraction (★) was excised and further analysed by ESI-MS.  (B)  SHuffle® T7 cells harbouring either pET23d(+):: HisDapGalNAcT2  in the absence or presence of pMJS9 were grown in EnPresso B medium. Fractionated cell samples were separated by SDS-PAGE and visualised by Coomassie staining  (B)  or immunoblotting  (C)  using a mouse anti human GALNT2 antibody. Molecular weight markers (MW) are in kDa. HisDapGalNAcT2 with an estimated mass of 61.7 kDa (arrow) was detected in soluble (S) and particulate (P) cell fractions. Commercially available rhGalNAcT2 (PC) and cell lysates of SHuffle® T7 pET23d(+) and SHuffle® T7 pMJS9 cells (NC) were included as controls.
    Figure Legend Snippet: SDS-PAGE and immunoblot analysis of HisDapGalNAcT2 expressed in E. coli . (A) Origami™ 2(DE3)pLysS cells carrying plasmid pET23d(+):: HisDapGalNAcT2 were grown in LB medium at 37°C until OD 600 0.5, at which point IPTG (final concentration 1 mM) was added and cultures were incubated for a further 5 h. Cells were harvested by centrifugation, lysed and total cell lysate (T), the soluble protein fraction (S) and the insoluble particulate fraction (P) were separated by SDS-PAGE and visualised by Coomassie staining. HisDapGalNAcT2 with an estimated mass of 61.7 kDa (indicated by arrow) was not detected in the soluble (S) cell fraction, but a band of the correct size in the insoluble particulate (P) fraction (★) was excised and further analysed by ESI-MS. (B) SHuffle® T7 cells harbouring either pET23d(+):: HisDapGalNAcT2 in the absence or presence of pMJS9 were grown in EnPresso B medium. Fractionated cell samples were separated by SDS-PAGE and visualised by Coomassie staining (B) or immunoblotting (C) using a mouse anti human GALNT2 antibody. Molecular weight markers (MW) are in kDa. HisDapGalNAcT2 with an estimated mass of 61.7 kDa (arrow) was detected in soluble (S) and particulate (P) cell fractions. Commercially available rhGalNAcT2 (PC) and cell lysates of SHuffle® T7 pET23d(+) and SHuffle® T7 pMJS9 cells (NC) were included as controls.

    Techniques Used: SDS Page, Plasmid Preparation, Concentration Assay, Incubation, Centrifugation, Staining, Mass Spectrometry, Molecular Weight

    5) Product Images from "The cysteine-rich exosporium morphogenetic protein, CdeC, exhibits self-assembly properties that lead to organized inclusion bodies in Escherichia coli"

    Article Title: The cysteine-rich exosporium morphogenetic protein, CdeC, exhibits self-assembly properties that lead to organized inclusion bodies in Escherichia coli

    Journal: bioRxiv

    doi: 10.1101/2020.07.09.196287

    Effect of recombinant strain type in the soluble expression and structural organization of CdeC. A) Recombinant CdeC expression in E. coli BL21 (DE3) pRIL and SHuffle T7 strains carrying pARR19 were induced with 0.5 mM IPTG for 16 h at 37°C. Cells were collected and lysed in soluble (S) and insoluble (I) lysis buffer, electrophoresed, and analyzed by Western blot as described in the Methods section. Each lane was loaded with 2 μg of protein lysate. Molecular mass (kDa) markers are indicated at the left side of the panels, and molecular mass of the detected His-tagged immunoreactive bands are indicated at the right side of the panels. B) Thin sections of E. coli BL21 (DE3) pRIL and E. coli SHuffle T7 expressing CdeC from C. difficile R20291 were analyzed by transmission electron microscopy as described in the Method section. Representative micrographs of several E. coli cells are shown in the upper panel. The middle panel shows selected individual cells, and the lower panel displays a magnified view of the thin section of inclusion bodies inside E. coli BL21 (DE3) pRIL or SHuffle T7. When corresponding, lamellae-like formation in CdeC is indicated with blue arrowheads.
    Figure Legend Snippet: Effect of recombinant strain type in the soluble expression and structural organization of CdeC. A) Recombinant CdeC expression in E. coli BL21 (DE3) pRIL and SHuffle T7 strains carrying pARR19 were induced with 0.5 mM IPTG for 16 h at 37°C. Cells were collected and lysed in soluble (S) and insoluble (I) lysis buffer, electrophoresed, and analyzed by Western blot as described in the Methods section. Each lane was loaded with 2 μg of protein lysate. Molecular mass (kDa) markers are indicated at the left side of the panels, and molecular mass of the detected His-tagged immunoreactive bands are indicated at the right side of the panels. B) Thin sections of E. coli BL21 (DE3) pRIL and E. coli SHuffle T7 expressing CdeC from C. difficile R20291 were analyzed by transmission electron microscopy as described in the Method section. Representative micrographs of several E. coli cells are shown in the upper panel. The middle panel shows selected individual cells, and the lower panel displays a magnified view of the thin section of inclusion bodies inside E. coli BL21 (DE3) pRIL or SHuffle T7. When corresponding, lamellae-like formation in CdeC is indicated with blue arrowheads.

    Techniques Used: Recombinant, Expressing, Lysis, Western Blot, Transmission Assay, Electron Microscopy

    6) Product Images from "The Clostridioides difficile Cysteine-Rich Exosporium Morphogenetic Protein, CdeC, Exhibits Self-Assembly Properties That Lead to Organized Inclusion Bodies in Escherichia coli"

    Article Title: The Clostridioides difficile Cysteine-Rich Exosporium Morphogenetic Protein, CdeC, Exhibits Self-Assembly Properties That Lead to Organized Inclusion Bodies in Escherichia coli

    Journal: mSphere

    doi: 10.1128/mSphere.01065-20

    Effect of recombinant strain type in the soluble expression and structural organization of CdeC. (A) Recombinant CdeC expression in E. coli BL21(DE3) pRIL and SHuffle T7 strains carrying pARR19 induced with 0.5 mM IPTG for 16 h at 37°C. Cells were collected and lysed in soluble (S) and insoluble (I) lysis buffers, electrophoresed, and analyzed by Western blotting as described in Materials and Methods. Each lane was loaded with 2 μg of protein lysat e . Molecular mass (kDa) markers are indicated at the left, and molecular mass of the detected His-tagged immunoreactive bands are indicated at the right. (B) Thin sections of E. coli BL21(DE3) pRIL and E. coli SHuffle T7 expressing CdeC from C. difficile R20291 were analyzed by transmission electron microscopy as described in Materials and Methods. (Top) Representative micrographs of several E. coli cells are shown. (Middle) Selected individual cells. (Bottom) Magnified views of the thin sections of inclusion bodies inside E. coli BL21(DE3) pRIL or SHuffle T7. Lamella-like formation in CdeC is indicated with blue arrowheads. Numbers in the black boxes indicate the percentages of cells showing a lamella-like structure.
    Figure Legend Snippet: Effect of recombinant strain type in the soluble expression and structural organization of CdeC. (A) Recombinant CdeC expression in E. coli BL21(DE3) pRIL and SHuffle T7 strains carrying pARR19 induced with 0.5 mM IPTG for 16 h at 37°C. Cells were collected and lysed in soluble (S) and insoluble (I) lysis buffers, electrophoresed, and analyzed by Western blotting as described in Materials and Methods. Each lane was loaded with 2 μg of protein lysat e . Molecular mass (kDa) markers are indicated at the left, and molecular mass of the detected His-tagged immunoreactive bands are indicated at the right. (B) Thin sections of E. coli BL21(DE3) pRIL and E. coli SHuffle T7 expressing CdeC from C. difficile R20291 were analyzed by transmission electron microscopy as described in Materials and Methods. (Top) Representative micrographs of several E. coli cells are shown. (Middle) Selected individual cells. (Bottom) Magnified views of the thin sections of inclusion bodies inside E. coli BL21(DE3) pRIL or SHuffle T7. Lamella-like formation in CdeC is indicated with blue arrowheads. Numbers in the black boxes indicate the percentages of cells showing a lamella-like structure.

    Techniques Used: Recombinant, Expressing, Lysis, Western Blot, Transmission Assay, Electron Microscopy

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  • 96
    New England Biolabs t7 shuffle
    Experiments to compare the yield of <t>T7</t> SHuffle ® , KGK10, BL21 DE3 Star, and the PURE frex 2.1 kit. KGK10 #1 is carefully fermented KGK10 extract that was provided by the Swartz lab. KGK10 #2 is grown in-house using a shake flask. (A) Gluc signal comparisons at a gain of 100 without PURE frex data (n=3). (B) Gluc signal comparisons with an instrument gain of 80 so PURE frex data does not saturate the detector. (C) Expression of sfGFP to compare yield of proteins without disulfide bonds over 16 hr (n=3). (D) Is a ratio of oxidation potential/productivity using a YFP-mCherry fusion where a S-S bond has been introduced into a YFP variant (n=3).
    T7 Shuffle, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 shuffle/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t7 shuffle - by Bioz Stars, 2022-08
    96/100 stars
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    93
    New England Biolabs shuffle strain c3030
    Cytoplamic expression of proTHI-TRX fusion proteins in strain <t>C3030.</t> For all fusion proteins, 1 ml of cell culture was pelleted and dissolved in 100 μl sample buffer. 10 μl from this extract and an equivalent amount for total soluble cytoplasmic fractions were separated on Tricine/SDS gels. (M) Protein marker, ( 1 ) un-induced crude extract, ( 2 ) induced crude extract, ( 3 ) total soluble fraction taken after cell lysis by sonication, ( 4 ) insoluble fraction after sonication. Red stars indicate the 25 kDa fusion protein
    Shuffle Strain C3030, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/shuffle strain c3030/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    shuffle strain c3030 - by Bioz Stars, 2022-08
    93/100 stars
      Buy from Supplier

    Image Search Results


    Experiments to compare the yield of T7 SHuffle ® , KGK10, BL21 DE3 Star, and the PURE frex 2.1 kit. KGK10 #1 is carefully fermented KGK10 extract that was provided by the Swartz lab. KGK10 #2 is grown in-house using a shake flask. (A) Gluc signal comparisons at a gain of 100 without PURE frex data (n=3). (B) Gluc signal comparisons with an instrument gain of 80 so PURE frex data does not saturate the detector. (C) Expression of sfGFP to compare yield of proteins without disulfide bonds over 16 hr (n=3). (D) Is a ratio of oxidation potential/productivity using a YFP-mCherry fusion where a S-S bond has been introduced into a YFP variant (n=3).

    Journal: bioRxiv

    Article Title: Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds

    doi: 10.1101/2019.12.19.883413

    Figure Lengend Snippet: Experiments to compare the yield of T7 SHuffle ® , KGK10, BL21 DE3 Star, and the PURE frex 2.1 kit. KGK10 #1 is carefully fermented KGK10 extract that was provided by the Swartz lab. KGK10 #2 is grown in-house using a shake flask. (A) Gluc signal comparisons at a gain of 100 without PURE frex data (n=3). (B) Gluc signal comparisons with an instrument gain of 80 so PURE frex data does not saturate the detector. (C) Expression of sfGFP to compare yield of proteins without disulfide bonds over 16 hr (n=3). (D) Is a ratio of oxidation potential/productivity using a YFP-mCherry fusion where a S-S bond has been introduced into a YFP variant (n=3).

    Article Snippet: To investigate this, we expressed sfGFP using BL21 DE3 Star, KGK10, and T7 SHuffle.

    Techniques: Expressing, Variant Assay

    Experiments to optimize expression from T7 Shuffle extract. (A) Response surface fit to determine optimal growth conditions for sfGFP expression; z axis shows fluorescence (B) Response surface fit to determine optimal growth conditions for Gluc expression; z axis shows luminescence.

    Journal: bioRxiv

    Article Title: Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds

    doi: 10.1101/2019.12.19.883413

    Figure Lengend Snippet: Experiments to optimize expression from T7 Shuffle extract. (A) Response surface fit to determine optimal growth conditions for sfGFP expression; z axis shows fluorescence (B) Response surface fit to determine optimal growth conditions for Gluc expression; z axis shows luminescence.

    Article Snippet: To investigate this, we expressed sfGFP using BL21 DE3 Star, KGK10, and T7 SHuffle.

    Techniques: Expressing, Fluorescence

    Schematic of mechanisms that affect disulfide bond formation and implications in the T7 Shuffle strain. (A) Oxidation via DsbA forms bonds between thiol groups on cysteines. (B) DsbC enzymes proofread proteins and isomerize disulfide bonds. (C) Reduction can occur via trxB and (D) gor enzymes that cleave disulfide bonds on the protein. (E) The T7 SHuffle ® strain is engineered to support disulfide bond formation by eliminating reducing enzymes and overexpressing the DsbC chaperone. This figure has been modified from a published schematic on disulfide bond formation ( Ke and Berkmen, 2014 ).

    Journal: bioRxiv

    Article Title: Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds

    doi: 10.1101/2019.12.19.883413

    Figure Lengend Snippet: Schematic of mechanisms that affect disulfide bond formation and implications in the T7 Shuffle strain. (A) Oxidation via DsbA forms bonds between thiol groups on cysteines. (B) DsbC enzymes proofread proteins and isomerize disulfide bonds. (C) Reduction can occur via trxB and (D) gor enzymes that cleave disulfide bonds on the protein. (E) The T7 SHuffle ® strain is engineered to support disulfide bond formation by eliminating reducing enzymes and overexpressing the DsbC chaperone. This figure has been modified from a published schematic on disulfide bond formation ( Ke and Berkmen, 2014 ).

    Article Snippet: To investigate this, we expressed sfGFP using BL21 DE3 Star, KGK10, and T7 SHuffle.

    Techniques: Modification

    Comparison of recombinant expression yields in E. coli and insect cells. Expression in S2 cells results in higher yields, purity and shows no correlation with construct length. ( A ) Size exclusion chromatograms and SDS-gel (inset) of shortest (dashed line) and longest (full line) ISG65 constructs expressed in E. coli and insect cells. Bacterial culture volumes were tenfold higher. The protein gel was digitally cropped for clarity. The complete gel is shown in Supplementary Figure 3 . ( B ) Expression yields normalised to 1 L of cell culture. Normalised yields were 38-fold (longest construct, dark grey), and, respectively, 250-fold higher (shortest construct, light grey) for the same constructs expressed in insect cells. EC, E. coli ; DM Drosophila melanogaster; M, Marker.

    Journal: Scientific Reports

    Article Title: A multifaceted strategy to improve recombinant expression and structural characterisation of a Trypanosoma invariant surface protein

    doi: 10.1038/s41598-022-16958-x

    Figure Lengend Snippet: Comparison of recombinant expression yields in E. coli and insect cells. Expression in S2 cells results in higher yields, purity and shows no correlation with construct length. ( A ) Size exclusion chromatograms and SDS-gel (inset) of shortest (dashed line) and longest (full line) ISG65 constructs expressed in E. coli and insect cells. Bacterial culture volumes were tenfold higher. The protein gel was digitally cropped for clarity. The complete gel is shown in Supplementary Figure 3 . ( B ) Expression yields normalised to 1 L of cell culture. Normalised yields were 38-fold (longest construct, dark grey), and, respectively, 250-fold higher (shortest construct, light grey) for the same constructs expressed in insect cells. EC, E. coli ; DM Drosophila melanogaster; M, Marker.

    Article Snippet: Expression constructs were introduced in E. coli T7 shuffle cells (New England Biolabs) by heat-shock transformation.

    Techniques: Recombinant, Expressing, Construct, SDS-Gel, Cell Culture, Marker

    Yields of E. coli expressed ISG65 decrease with construct length. ( A ) Stacked size exclusion chromatograms of all ISG65 constructs expressed in E. coli . Major tick marks correspond to 300 mAU. Numbers indicate the position of the C-terminal truncation. Peaks corresponding to correctly folded ISG65 are indicated by a black horizontal bar, aggregates by an arrow. The void volume of the column is marked by a dashed, vertical line. ( B ) SDS-PAGE analysis of SEC peak fractions. Total protein yields were calculated for each peak by UV 280nm absorption and were normalized to the cell mass in 1 L of culture. All edges of the gel, except the left side, are digitally cropped for clarity. Displayed is the whole separating gel as indicated by the lowest and the highest molecular weight bands of the marker (M).

    Journal: Scientific Reports

    Article Title: A multifaceted strategy to improve recombinant expression and structural characterisation of a Trypanosoma invariant surface protein

    doi: 10.1038/s41598-022-16958-x

    Figure Lengend Snippet: Yields of E. coli expressed ISG65 decrease with construct length. ( A ) Stacked size exclusion chromatograms of all ISG65 constructs expressed in E. coli . Major tick marks correspond to 300 mAU. Numbers indicate the position of the C-terminal truncation. Peaks corresponding to correctly folded ISG65 are indicated by a black horizontal bar, aggregates by an arrow. The void volume of the column is marked by a dashed, vertical line. ( B ) SDS-PAGE analysis of SEC peak fractions. Total protein yields were calculated for each peak by UV 280nm absorption and were normalized to the cell mass in 1 L of culture. All edges of the gel, except the left side, are digitally cropped for clarity. Displayed is the whole separating gel as indicated by the lowest and the highest molecular weight bands of the marker (M).

    Article Snippet: Expression constructs were introduced in E. coli T7 shuffle cells (New England Biolabs) by heat-shock transformation.

    Techniques: Construct, SDS Page, Molecular Weight, Marker

    Experiments to compare the yield of T7 SHuffle ® , KGK10, BL21 DE3 Star, and the PURE frex 2.1 kit. KGK10 #1 is carefully fermented KGK10 extract that was provided by the Swartz lab. KGK10 #2 is grown in-house using a shake flask. (A) Gluc signal comparisons at a gain of 100 without PURE frex data (n=3). (B) Gluc signal comparisons with an instrument gain of 80 so PURE frex data does not saturate the detector. (C) Expression of sfGFP to compare yield of proteins without disulfide bonds over 16 hr (n=3). (D) Is a ratio of oxidation potential/productivity using a YFP-mCherry fusion where a S-S bond has been introduced into a YFP variant (n=3).

    Journal: bioRxiv

    Article Title: Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds

    doi: 10.1101/2019.12.19.883413

    Figure Lengend Snippet: Experiments to compare the yield of T7 SHuffle ® , KGK10, BL21 DE3 Star, and the PURE frex 2.1 kit. KGK10 #1 is carefully fermented KGK10 extract that was provided by the Swartz lab. KGK10 #2 is grown in-house using a shake flask. (A) Gluc signal comparisons at a gain of 100 without PURE frex data (n=3). (B) Gluc signal comparisons with an instrument gain of 80 so PURE frex data does not saturate the detector. (C) Expression of sfGFP to compare yield of proteins without disulfide bonds over 16 hr (n=3). (D) Is a ratio of oxidation potential/productivity using a YFP-mCherry fusion where a S-S bond has been introduced into a YFP variant (n=3).

    Article Snippet: To investigate this, we expressed sfGFP using BL21 DE3 Star, KGK10, and T7 SHuffle.

    Techniques: Expressing, Variant Assay

    Experiments to optimize expression from T7 Shuffle extract. (A) Response surface fit to determine optimal growth conditions for sfGFP expression; z axis shows fluorescence (B) Response surface fit to determine optimal growth conditions for Gluc expression; z axis shows luminescence.

    Journal: bioRxiv

    Article Title: Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds

    doi: 10.1101/2019.12.19.883413

    Figure Lengend Snippet: Experiments to optimize expression from T7 Shuffle extract. (A) Response surface fit to determine optimal growth conditions for sfGFP expression; z axis shows fluorescence (B) Response surface fit to determine optimal growth conditions for Gluc expression; z axis shows luminescence.

    Article Snippet: To investigate this, we expressed sfGFP using BL21 DE3 Star, KGK10, and T7 SHuffle.

    Techniques: Expressing, Fluorescence

    Schematic of mechanisms that affect disulfide bond formation and implications in the T7 Shuffle strain. (A) Oxidation via DsbA forms bonds between thiol groups on cysteines. (B) DsbC enzymes proofread proteins and isomerize disulfide bonds. (C) Reduction can occur via trxB and (D) gor enzymes that cleave disulfide bonds on the protein. (E) The T7 SHuffle ® strain is engineered to support disulfide bond formation by eliminating reducing enzymes and overexpressing the DsbC chaperone. This figure has been modified from a published schematic on disulfide bond formation ( Ke and Berkmen, 2014 ).

    Journal: bioRxiv

    Article Title: Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds

    doi: 10.1101/2019.12.19.883413

    Figure Lengend Snippet: Schematic of mechanisms that affect disulfide bond formation and implications in the T7 Shuffle strain. (A) Oxidation via DsbA forms bonds between thiol groups on cysteines. (B) DsbC enzymes proofread proteins and isomerize disulfide bonds. (C) Reduction can occur via trxB and (D) gor enzymes that cleave disulfide bonds on the protein. (E) The T7 SHuffle ® strain is engineered to support disulfide bond formation by eliminating reducing enzymes and overexpressing the DsbC chaperone. This figure has been modified from a published schematic on disulfide bond formation ( Ke and Berkmen, 2014 ).

    Article Snippet: To investigate this, we expressed sfGFP using BL21 DE3 Star, KGK10, and T7 SHuffle.

    Techniques: Modification

    Cytoplamic expression of proTHI-TRX fusion proteins in strain C3030. For all fusion proteins, 1 ml of cell culture was pelleted and dissolved in 100 μl sample buffer. 10 μl from this extract and an equivalent amount for total soluble cytoplasmic fractions were separated on Tricine/SDS gels. (M) Protein marker, ( 1 ) un-induced crude extract, ( 2 ) induced crude extract, ( 3 ) total soluble fraction taken after cell lysis by sonication, ( 4 ) insoluble fraction after sonication. Red stars indicate the 25 kDa fusion protein

    Journal: Biotechnology Letters

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli

    doi: 10.1007/s10529-013-1180-z

    Figure Lengend Snippet: Cytoplamic expression of proTHI-TRX fusion proteins in strain C3030. For all fusion proteins, 1 ml of cell culture was pelleted and dissolved in 100 μl sample buffer. 10 μl from this extract and an equivalent amount for total soluble cytoplasmic fractions were separated on Tricine/SDS gels. (M) Protein marker, ( 1 ) un-induced crude extract, ( 2 ) induced crude extract, ( 3 ) total soluble fraction taken after cell lysis by sonication, ( 4 ) insoluble fraction after sonication. Red stars indicate the 25 kDa fusion protein

    Article Snippet: The amount of fusion protein that could be produced from the SHuffle strain C3030 is shown in Table and was higher than obtained with Rosetta(DE3)pLysS.

    Techniques: Expressing, Cell Culture, Marker, Lysis, Sonication

    Comparison of Ni–NTA purified proTHI-TRX fusion proteins from strain C3030. a Coomassie Brilliant Blue staining. b Western blot with anti-His tag antibody. Each well contained 3 μg protein. (M) Prestained protein marker, ( 1 ) proTHI2.1-TRX, ( 2 ) proTHI2.2-TRX, ( 3 ) proTHI2.3-TRX, ( 4 ) proTHI2.4-TRX

    Journal: Biotechnology Letters

    Article Title: Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli

    doi: 10.1007/s10529-013-1180-z

    Figure Lengend Snippet: Comparison of Ni–NTA purified proTHI-TRX fusion proteins from strain C3030. a Coomassie Brilliant Blue staining. b Western blot with anti-His tag antibody. Each well contained 3 μg protein. (M) Prestained protein marker, ( 1 ) proTHI2.1-TRX, ( 2 ) proTHI2.2-TRX, ( 3 ) proTHI2.3-TRX, ( 4 ) proTHI2.4-TRX

    Article Snippet: The amount of fusion protein that could be produced from the SHuffle strain C3030 is shown in Table and was higher than obtained with Rosetta(DE3)pLysS.

    Techniques: Purification, Staining, Western Blot, Marker