t7 shuffle  (New England Biolabs)


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    Structured Review

    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
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    Average 96 stars, based on 1 article reviews
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    t7 shuffle - by Bioz Stars, 2022-05
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    Images

    1) Product Images from "Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds"

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

    Journal: bioRxiv

    doi: 10.1101/2019.12.19.883413

    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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 ).
    Figure Legend 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 ).

    Techniques Used: Modification

    2) Product Images from "Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds"

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

    Journal: bioRxiv

    doi: 10.1101/2019.12.19.883413

    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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 ).
    Figure Legend 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 ).

    Techniques Used: Modification

    3) 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

    4) Product Images from "Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds"

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

    Journal: bioRxiv

    doi: 10.1101/2019.12.19.883413

    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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 ).
    Figure Legend 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 ).

    Techniques Used: Modification

    5) Product Images from "Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A"

    Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

    Journal: bioRxiv

    doi: 10.1101/2021.01.31.429007

    Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. A. RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). B. Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. C. Ribonuclease activity assay on dsRNA.
    Figure Legend Snippet: Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. A. RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). B. Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. C. Ribonuclease activity assay on dsRNA.

    Techniques Used: Expressing, Purification, Activity Assay, Column Chromatography, Magnetic Beads

    Comparison of protein expression in three E. coli strains: NEB Turbo, NEB Express, T7 SHuffle (K strain). MBP-ACE2NTD (ACE), MBP-TMPRSS2 (PRS, lacking the transmembrane domain), MBP-RNase I (RI), MBP-RNase A (RA). A. SDS-PAGE analysis of total proteins in cell lysate. B. SDS-PAGE analysis of soluble proteins (supernatant) in cell lysate. “*” indicates the expected target protein.
    Figure Legend Snippet: Comparison of protein expression in three E. coli strains: NEB Turbo, NEB Express, T7 SHuffle (K strain). MBP-ACE2NTD (ACE), MBP-TMPRSS2 (PRS, lacking the transmembrane domain), MBP-RNase I (RI), MBP-RNase A (RA). A. SDS-PAGE analysis of total proteins in cell lysate. B. SDS-PAGE analysis of soluble proteins (supernatant) in cell lysate. “*” indicates the expected target protein.

    Techniques Used: Expressing, SDS Page

    6) Product Images from "Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A"

    Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.660149

    Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. (A) RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026, K strain), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). (B) Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. (C) Ribonuclease activity assay on dsRNA (40 mer duplex and dsRNA ladder).
    Figure Legend Snippet: Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. (A) RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026, K strain), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). (B) Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. (C) Ribonuclease activity assay on dsRNA (40 mer duplex and dsRNA ladder).

    Techniques Used: Expressing, Purification, Activity Assay, Column Chromatography, Magnetic Beads

    7) Product Images from "Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds"

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

    Journal: bioRxiv

    doi: 10.1101/2019.12.19.883413

    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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 ).
    Figure Legend 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 ).

    Techniques Used: Modification

    8) Product Images from "Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains"

    Article Title: Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains

    Journal: Research in Pharmaceutical Sciences

    doi: 10.4103/1735-5362.288424

    GM-CSF expression in  Escherichia coli  Shuffle T7. Protein expression was induced with (A) 1 mM and (B) 0.5 mM isopropyl-β-D-thiogalactopyranoside. Insoluble (lanes 1, 3, and 5) and soluble (lanes 2, 4, and 6) fractions of protein expresed at 37 °C, 30 °C and 23 °C, respectivly. Lane M, Molecular weight protein marker and lane U, uniduced sample. GM-CSF (15 kDa) is indicated by arrow.
    Figure Legend Snippet: GM-CSF expression in Escherichia coli Shuffle T7. Protein expression was induced with (A) 1 mM and (B) 0.5 mM isopropyl-β-D-thiogalactopyranoside. Insoluble (lanes 1, 3, and 5) and soluble (lanes 2, 4, and 6) fractions of protein expresed at 37 °C, 30 °C and 23 °C, respectivly. Lane M, Molecular weight protein marker and lane U, uniduced sample. GM-CSF (15 kDa) is indicated by arrow.

    Techniques Used: Expressing, Molecular Weight, Marker

    9) 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

    10) Product Images from "Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds"

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

    Journal: bioRxiv

    doi: 10.1101/2019.12.19.883413

    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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 ).
    Figure Legend 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 ).

    Techniques Used: Modification

    11) Product Images from "Simple, Functional, Inexpensive Cell Extract for in vitro Prototyping of Proteins with Disulfide Bonds"

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

    Journal: bioRxiv

    doi: 10.1101/2019.12.19.883413

    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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 ).
    Figure Legend 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 ).

    Techniques Used: Modification

    12) 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

    13) Product Images from "Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A"

    Article Title: Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.660149

    Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. (A) RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026, K strain), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). (B) Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. (C) Ribonuclease activity assay on dsRNA (40 mer duplex and dsRNA ladder).
    Figure Legend Snippet: Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. (A) RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026, K strain), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). (B) Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. (C) Ribonuclease activity assay on dsRNA (40 mer duplex and dsRNA ladder).

    Techniques Used: Expressing, Purification, Activity Assay, Column Chromatography, Magnetic Beads

    14) Product Images from "An unusual thioredoxin system in the facultative parasite Acanthamoeba castellanii"

    Article Title: An unusual thioredoxin system in the facultative parasite Acanthamoeba castellanii

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-021-03786-x

    Intracellular localization of the three disulfide reductases in A . castellanii Neff. a Western blots of fractions obtained through subcellular fractionation of A . castellanii cell extracts with purified antibodies (dilution 1:100) against either disulfide reductase. The loaded amount of protein was 100 µg for both, supernatant (SN) and the heavy fraction from a Percoll gradient (HF) [ 42 ]. The additional bands in the pellet fraction obtained with α-Ac GR-antibody might refer to alternatively spliced and partially degraded Ac GR. The arrow indicates the position of Ac TrxR-S. The thin black lines separating the lanes indicate that samples were run together on one gel and blotted together on a PVDF membrane, but that the blots were developed independently. b Western blot with α-Ac TrxR-S (dilution 1:100) of supernatant (SN) and pelleted fractions (PF), obtained by centrifugation (4 °C, 20,000 × g, 10 min) of cell lysates of A . castellanii Neff cells after treatment with 750 µM H 2 O 2 for 18 h. The latter fraction contains all larger organelles. c Immunofluorescence images (1000 × magnification) of A . castellanii Neff cells after staining with purified antibodies (dilution 1:200) against each of the disulfide reductases. Secondary antibodies (dilutions 1: 10,000) were either conjugated with Alexa Fluor 488 (rabbit anti-Mouse IgG for Ac TrxR-L) or with TRITC (goat anti-rabbit IgG for Ac TrxR-S and Ac GR). Nuclei were stained with DAPI (blue). Light microscopic images of the same cells are given below the according fluorescence images. Scale bar = 5 µm
    Figure Legend Snippet: Intracellular localization of the three disulfide reductases in A . castellanii Neff. a Western blots of fractions obtained through subcellular fractionation of A . castellanii cell extracts with purified antibodies (dilution 1:100) against either disulfide reductase. The loaded amount of protein was 100 µg for both, supernatant (SN) and the heavy fraction from a Percoll gradient (HF) [ 42 ]. The additional bands in the pellet fraction obtained with α-Ac GR-antibody might refer to alternatively spliced and partially degraded Ac GR. The arrow indicates the position of Ac TrxR-S. The thin black lines separating the lanes indicate that samples were run together on one gel and blotted together on a PVDF membrane, but that the blots were developed independently. b Western blot with α-Ac TrxR-S (dilution 1:100) of supernatant (SN) and pelleted fractions (PF), obtained by centrifugation (4 °C, 20,000 × g, 10 min) of cell lysates of A . castellanii Neff cells after treatment with 750 µM H 2 O 2 for 18 h. The latter fraction contains all larger organelles. c Immunofluorescence images (1000 × magnification) of A . castellanii Neff cells after staining with purified antibodies (dilution 1:200) against each of the disulfide reductases. Secondary antibodies (dilutions 1: 10,000) were either conjugated with Alexa Fluor 488 (rabbit anti-Mouse IgG for Ac TrxR-L) or with TRITC (goat anti-rabbit IgG for Ac TrxR-S and Ac GR). Nuclei were stained with DAPI (blue). Light microscopic images of the same cells are given below the according fluorescence images. Scale bar = 5 µm

    Techniques Used: Western Blot, Fractionation, Purification, Centrifugation, Immunofluorescence, Staining, Fluorescence

    Expression of Ac GR in G . lamblia . Recombinant Ac GR was expressed in G . lamblia with the pPac-VInteg vector [ 31 ] and isolated in Ni-NTA agarose columns via its C-terminal 6 × histidine tag. The left panel shows a western blot with 10 µg of eluate and a polyclonal α-Ac GR serum (rabbit; dilution 1:2000). The predicted size of Ac GR based on its 454 aa and the 6 × histidine tag is approximately 49 kDa. The right panel shows Ac GR activity in transfected G . lamblia (mean and SD from 11 measurements) and in wild-type A . castellanii Neff (mean and SD from six measurements). Measurements were done at λ = 412 in 100 mM potassium phosphate buffer with 0.2 mM NADPH, 1 mM DTNB, 1 mM GSSG, and cell extract at the concentration of 50 µg protein ml −1 . Values were read as reduction of DTNB by GSH, previously formed through reduction of GSSG by GSH
    Figure Legend Snippet: Expression of Ac GR in G . lamblia . Recombinant Ac GR was expressed in G . lamblia with the pPac-VInteg vector [ 31 ] and isolated in Ni-NTA agarose columns via its C-terminal 6 × histidine tag. The left panel shows a western blot with 10 µg of eluate and a polyclonal α-Ac GR serum (rabbit; dilution 1:2000). The predicted size of Ac GR based on its 454 aa and the 6 × histidine tag is approximately 49 kDa. The right panel shows Ac GR activity in transfected G . lamblia (mean and SD from 11 measurements) and in wild-type A . castellanii Neff (mean and SD from six measurements). Measurements were done at λ = 412 in 100 mM potassium phosphate buffer with 0.2 mM NADPH, 1 mM DTNB, 1 mM GSSG, and cell extract at the concentration of 50 µg protein ml −1 . Values were read as reduction of DTNB by GSH, previously formed through reduction of GSSG by GSH

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Isolation, Western Blot, Activity Assay, Transfection, Concentration Assay

    Recombinant expression of Ac TrxR-L in A . castellanii Neff. 2D-gels of A . castellanii Neff: cells were transfected with expression plasmids either carrying the Ac TrxR-L gene with an intact 3′UTR and an N-terminal 6 × His tag ( b ), an 3′ UTR with a scrambled SECIS ( c ) and an N-terminal 6 × His tag, or a truncated TrxR-L gene terminating at the UGA without an N-terminal 6 × His tag ( d ). a , 2D-gel of Neff control cells. Depictions of the gene constructs are given below the respective 2D-gels. Red rectangles indicate recombinant Ac TrxR-L without the terminal selenocysteine as expressed in transfected cell lines. Ac TrxR-L with 6 × His tag migrates towards a higher pI due to the positive charge of the additional histidines. The blue rectangle indicates the position of wild-type Ac TrxR-L in normal Neff cells
    Figure Legend Snippet: Recombinant expression of Ac TrxR-L in A . castellanii Neff. 2D-gels of A . castellanii Neff: cells were transfected with expression plasmids either carrying the Ac TrxR-L gene with an intact 3′UTR and an N-terminal 6 × His tag ( b ), an 3′ UTR with a scrambled SECIS ( c ) and an N-terminal 6 × His tag, or a truncated TrxR-L gene terminating at the UGA without an N-terminal 6 × His tag ( d ). a , 2D-gel of Neff control cells. Depictions of the gene constructs are given below the respective 2D-gels. Red rectangles indicate recombinant Ac TrxR-L without the terminal selenocysteine as expressed in transfected cell lines. Ac TrxR-L with 6 × His tag migrates towards a higher pI due to the positive charge of the additional histidines. The blue rectangle indicates the position of wild-type Ac TrxR-L in normal Neff cells

    Techniques Used: Recombinant, Expressing, Transfection, Two-Dimensional Gel Electrophoresis, Construct

    15) 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

    16) 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

    17) Product Images from "Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A"

    Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

    Journal: bioRxiv

    doi: 10.1101/2021.01.31.429007

    Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. A. RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). B. Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. C. Ribonuclease activity assay on dsRNA.
    Figure Legend Snippet: Expression and purification of E. coli RNase III and RNase activity assay on dsRNA. A. RNase III expression level in three E. coli T7 strains: T7 Shuffle (C3026), T7 Express with lacI q and LysY (C3013), and Nico (λDE3). B. Purified RNase III from nickel-NTA agarose column chromatography and Ni magnetic beads. C. Ribonuclease activity assay on dsRNA.

    Techniques Used: Expressing, Purification, Activity Assay, Column Chromatography, Magnetic Beads

    Comparison of protein expression in three E. coli strains: NEB Turbo, NEB Express, T7 SHuffle (K strain). MBP-ACE2NTD (ACE), MBP-TMPRSS2 (PRS, lacking the transmembrane domain), MBP-RNase I (RI), MBP-RNase A (RA). A. SDS-PAGE analysis of total proteins in cell lysate. B. SDS-PAGE analysis of soluble proteins (supernatant) in cell lysate. “*” indicates the expected target protein.
    Figure Legend Snippet: Comparison of protein expression in three E. coli strains: NEB Turbo, NEB Express, T7 SHuffle (K strain). MBP-ACE2NTD (ACE), MBP-TMPRSS2 (PRS, lacking the transmembrane domain), MBP-RNase I (RI), MBP-RNase A (RA). A. SDS-PAGE analysis of total proteins in cell lysate. B. SDS-PAGE analysis of soluble proteins (supernatant) in cell lysate. “*” indicates the expected target protein.

    Techniques Used: Expressing, SDS Page

    18) 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

    19) Product Images from "N-glycosylation on Oryza Sativa Root Germin-like Protein 1 is conserved but not required for stability or activity"

    Article Title: N-glycosylation on Oryza Sativa Root Germin-like Protein 1 is conserved but not required for stability or activity

    Journal: bioRxiv

    doi: 10.1101/2021.02.09.430526

    Os RGLP1 purified from K12 shuffle E. coli with MgATP wash has thermostable SOD activity. (A) SDS-PAGE of Os RGLP1 and co-purified E. coli GroL from K12 shuffle E. coli grown at different temperatures. (B) SDS-PAGE of Os RGLP1 purified without and with additional MgATP and misfolded bacterial protein extract wash. (C) Intact mass analysis of His-tagged Os RGLP1 purified from E. coli . Deconvoluted spectrum showing neutral average mass. (D) In-gel SOD activity assay (SOD activity appears as clear bands) and ( E ) replicate Coomassie stained seminative PAGE, SOD activity appears as clear bands in (D). (F) Quantitation of in-gel SOD activity. Values, mean. Error bars, S.D. *, p
    Figure Legend Snippet: Os RGLP1 purified from K12 shuffle E. coli with MgATP wash has thermostable SOD activity. (A) SDS-PAGE of Os RGLP1 and co-purified E. coli GroL from K12 shuffle E. coli grown at different temperatures. (B) SDS-PAGE of Os RGLP1 purified without and with additional MgATP and misfolded bacterial protein extract wash. (C) Intact mass analysis of His-tagged Os RGLP1 purified from E. coli . Deconvoluted spectrum showing neutral average mass. (D) In-gel SOD activity assay (SOD activity appears as clear bands) and ( E ) replicate Coomassie stained seminative PAGE, SOD activity appears as clear bands in (D). (F) Quantitation of in-gel SOD activity. Values, mean. Error bars, S.D. *, p

    Techniques Used: Purification, Activity Assay, SDS Page, Staining, Polyacrylamide Gel Electrophoresis, Quantitation Assay

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    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
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    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

    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

    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.

    Journal: Microbes and Environments

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

    doi: 10.1264/jsme2.ME19086

    Figure Lengend 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.

    Article Snippet: Escherichia coli strains DH5α (Invitrogen) and SHuffle T7 (New England Biolabs) were grown in LB medium supplemented where necessary with kanamycin (30‍ ‍μg‍ ‍mL‍–1 ) and were then incubated with shaking (250 rpm) at 37°C.

    Techniques: 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.

    Journal: Microbes and Environments

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

    doi: 10.1264/jsme2.ME19086

    Figure Lengend 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.

    Article Snippet: Escherichia coli strains DH5α (Invitrogen) and SHuffle T7 (New England Biolabs) were grown in LB medium supplemented where necessary with kanamycin (30‍ ‍μg‍ ‍mL‍–1 ) and were then incubated with shaking (250 rpm) at 37°C.

    Techniques: 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.

    Journal: Microbes and Environments

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

    doi: 10.1264/jsme2.ME19086

    Figure Lengend 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.

    Article Snippet: Escherichia coli strains DH5α (Invitrogen) and SHuffle T7 (New England Biolabs) were grown in LB medium supplemented where necessary with kanamycin (30‍ ‍μg‍ ‍mL‍–1 ) and were then incubated with shaking (250 rpm) at 37°C.

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