shuffle t7 express  (New England Biolabs)


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    New England Biolabs shuffle t7 express
    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 <t>T7</t> Express was required for soluble expression of some orthologs.
    Shuffle T7 Express, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) 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

    2) Product Images from "Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host"

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    Journal: BMC Biochemistry

    doi: 10.1186/s12858-018-0101-0

    Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification
    Figure Legend Snippet: Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Staining, Fluorescence, Purification

    Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa
    Figure Legend Snippet: Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    Techniques Used: Expressing, Recombinant, SDS Page, Over Expression

    Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)
    Figure Legend Snippet: Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Western Blot

    3) Product Images from "Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host"

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    Journal: BMC Biochemistry

    doi: 10.1186/s12858-018-0101-0

    Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification
    Figure Legend Snippet: Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Staining, Fluorescence, Purification

    Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa
    Figure Legend Snippet: Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    Techniques Used: Expressing, Recombinant, SDS Page, Over Expression

    Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)
    Figure Legend Snippet: Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Western Blot

    4) Product Images from "Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli"

    Article Title: Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli

    Journal: bioRxiv

    doi: 10.1101/2020.05.09.085944

    Phenotypic selection of cyclonal variants with differential antigen-binding activity. (a) Representative selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding spTorA-CAT-Gcn4-PP and a second plasmid encoding anti-Gcn4 cyclonal parent (GLF) or variant with CDR-H3 mutation as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM IPTG to induce protein expression. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate. (b) Survival curves for serially diluted SHuffle T7 Express cells co-expressing an anti-Gcn4 cyclonal variant along with the spTorA-CAT-Gcn4-PP reporter. Cells expressing the parental GLF cyclonal along with the non-cognate spTorA-CAT-HAG chimeric antigen (open circle) served as a negative control. Overnight cultures were serially diluted in liquid LB and plated on LB-agar supplemented with Cm. Maximal cell dilution that allowed growth is plotted versus Cm concentration. Arrow in (b) indicates data depicted in image panel (a) and corresponds to 20 μg/ml Cm.
    Figure Legend Snippet: Phenotypic selection of cyclonal variants with differential antigen-binding activity. (a) Representative selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding spTorA-CAT-Gcn4-PP and a second plasmid encoding anti-Gcn4 cyclonal parent (GLF) or variant with CDR-H3 mutation as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM IPTG to induce protein expression. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate. (b) Survival curves for serially diluted SHuffle T7 Express cells co-expressing an anti-Gcn4 cyclonal variant along with the spTorA-CAT-Gcn4-PP reporter. Cells expressing the parental GLF cyclonal along with the non-cognate spTorA-CAT-HAG chimeric antigen (open circle) served as a negative control. Overnight cultures were serially diluted in liquid LB and plated on LB-agar supplemented with Cm. Maximal cell dilution that allowed growth is plotted versus Cm concentration. Arrow in (b) indicates data depicted in image panel (a) and corresponds to 20 μg/ml Cm.

    Techniques Used: Selection, Binding Assay, Activity Assay, Plasmid Preparation, Variant Assay, Mutagenesis, Expressing, Negative Control, Concentration Assay

    Schematic of Tat-dependent positive selection for antigen-binding activity of full-length IgGs. In the absence of a cognate binding protein, a chimeric antigen comprised of a peptide or protein antigen of interest (orange) fused to the C-terminus of spTorA-CAT (blue) is exported out of the cytoplasm by the TatABC translocase. By localizing the chimeric antigen into the periplasm, the CAT domain is no longer able to inactivate the antibiotic chloramphenicol (Cm) by acetylation using acetyl-coenzyme A (CoA) and the host cells are rendered sensitive to antibiotic. When a cyclonal (yellow/black) is functionally expressed in the cytoplasm of SHuffle T7 Express cells, it binds specifically to the chimeric antigen, thereby sequestering the CAT domain in the cytoplasm where it can efficiently detoxify Cm (green star) and conferring an antibiotic-resistant phenotype. Individual clones from the selection plate are selected, genetically identified, and functionally characterized. Yellow balls/sticks represent the 16 intra- and intermolecular disulfide bonds in IgG that are required for folding and activity. (b) Schematic of pBAD24-based vector for expression of chimeric antigen constructs (left) and pCD1-based vector for expression of cyclonal IgGs (right). Abbreviations: RBS, ribosome-binding site; sp, TorA signal peptide; L, flexible GTSAAAG linker; Ag, antigen; V H , variable heavy; V L , variable light; CH, constant heavy; CL, constant light.
    Figure Legend Snippet: Schematic of Tat-dependent positive selection for antigen-binding activity of full-length IgGs. In the absence of a cognate binding protein, a chimeric antigen comprised of a peptide or protein antigen of interest (orange) fused to the C-terminus of spTorA-CAT (blue) is exported out of the cytoplasm by the TatABC translocase. By localizing the chimeric antigen into the periplasm, the CAT domain is no longer able to inactivate the antibiotic chloramphenicol (Cm) by acetylation using acetyl-coenzyme A (CoA) and the host cells are rendered sensitive to antibiotic. When a cyclonal (yellow/black) is functionally expressed in the cytoplasm of SHuffle T7 Express cells, it binds specifically to the chimeric antigen, thereby sequestering the CAT domain in the cytoplasm where it can efficiently detoxify Cm (green star) and conferring an antibiotic-resistant phenotype. Individual clones from the selection plate are selected, genetically identified, and functionally characterized. Yellow balls/sticks represent the 16 intra- and intermolecular disulfide bonds in IgG that are required for folding and activity. (b) Schematic of pBAD24-based vector for expression of chimeric antigen constructs (left) and pCD1-based vector for expression of cyclonal IgGs (right). Abbreviations: RBS, ribosome-binding site; sp, TorA signal peptide; L, flexible GTSAAAG linker; Ag, antigen; V H , variable heavy; V L , variable light; CH, constant heavy; CL, constant light.

    Techniques Used: Selection, Binding Assay, Activity Assay, Clone Assay, Plasmid Preparation, Expressing, Construct

    Genetic selection for cyclonal antigen-binding activity. Selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding one of the chimeric antigens (spTorA-CAT-Ag or an export-defective variant spTorA(KK)-CAT-Ag) alone or with a second plasmid encoding a full-length cyclonal IgG specific for HAG, Gcn4-PP, or c-Myc as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) to induce chimeric antigen and cyclonal expression, respectively. Cross-pairing the anti-HAG cyclonal with non-cognate c-Myc or Gcn4-PP and the anti-Gcn4 cyclonal with non-cognate HAG served as negative controls. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate.
    Figure Legend Snippet: Genetic selection for cyclonal antigen-binding activity. Selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding one of the chimeric antigens (spTorA-CAT-Ag or an export-defective variant spTorA(KK)-CAT-Ag) alone or with a second plasmid encoding a full-length cyclonal IgG specific for HAG, Gcn4-PP, or c-Myc as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) to induce chimeric antigen and cyclonal expression, respectively. Cross-pairing the anti-HAG cyclonal with non-cognate c-Myc or Gcn4-PP and the anti-Gcn4 cyclonal with non-cognate HAG served as negative controls. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate.

    Techniques Used: Selection, Binding Assay, Activity Assay, Plasmid Preparation, Variant Assay, Expressing

    5) Product Images from "Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm"

    Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm

    Journal: Biotechnology progress

    doi: 10.1002/btpr.3102

    (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value
    Figure Legend Snippet: (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value

    Techniques Used: Western Blot, Expressing, Recombinant, Produced, Bradford Assay, Software, Standard Deviation

    (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.
    Figure Legend Snippet: (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.

    Techniques Used: Western Blot, Expressing, Binding Assay, Activity Assay, Purification, Construct, Standard Deviation

    SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.
    Figure Legend Snippet: SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.

    Techniques Used: SDS Page, Construct, Purification, Molecular Weight, Marker

    6) Product Images from "A Hetero-Multimeric Chitinase-Containing Plasmodium falciparum and Plasmodium gallinaceum Ookinete-Secreted Protein Complex Involved in Mosquito Midgut Invasion"

    Article Title: A Hetero-Multimeric Chitinase-Containing Plasmodium falciparum and Plasmodium gallinaceum Ookinete-Secreted Protein Complex Involved in Mosquito Midgut Invasion

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2020.615343

    Site-directed mutagenesis of conserved cysteines located in the chitin binding domain of P. gallinaceum chitinase ( rPgCHT1 ) and evaluation of chitin binding affinity of mutant rPgCHT1 and rPfCHT1 . Three conserved three cysteines (A) that were selected for mutation using the QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene) with the known template ( PgCHT1 - PET32b plasmid DNA expression construct) containing a thioredoxin epitope tag (TRX-Tag). The changes of single base pairs were achieved within codons 452 aa (1,355 bp, TGC to TCC), 501 aa (1,502 bp, TGC to TCC) and 557 aa (1,670 bp, TGT to TCT) of the plasmid constructs to mutate cysteine (C) residues to serine (S) as shown in (B) . The PfCHT1 - pET-32b expression plasmid was obtained from Gene Universal (Newark DE). The #1 mutant plasmid (in which all three C were modified), #2 plasmid (only two C modified), and wild type (3, rPgCHT2 -PET32) and rPfCHT1 were transformed into SHuffle T7 Express Competent E. coli cells for protein expression. The rPgCHT1 lysates were incubated with chitin beads, washed three times either with either PBS (1, 2, 3) or PBS with 1% Triton X-100 (1-Tx, 2-Tx, and 3-Tx) to remove non-specific or loosely bound chitinase, and evaluated for binding affinity of the mutant rPgCHT1. Similarly, primary E. coli lysate containing rPfCHT1 in soluble form Ni-NTA purified rPfCHT1 was incubated with chitin beads and washed with PBS and PBST (0.2%–1% Triton-X 100). The bound chitin beads were mixed with sample buffer and subjected to SDS-PAGE followed by Western immunoblot analysis with anti-thioredoxin monoclonal antibody (Abcam) to detect rPgCHT1, and 1C3 antibody to detect rPfCHT1 (E, F) . Panel (C) shows the expression of rPgCHT1 (75 kDa) protein in all clones. The binding affinity to chitin beads of mutant #58 (all three cysteines modified) was lower and largely eliminated with the Triton-X 100 wash (D) , indicating that the chitin binding domain is essential for the interaction of the long form chitinase ( PgCHT1 ) to chitin. Panels (E, F) show the strong binding of rPfCHT1 to chitin beads in both (E) coli culture lysates and purified rPfCHT1. In (E, F) , chitin beads were washed with PBS with different concentrations of Triton X 100 (0%–1%). rPfCHT1 strongly binds to chitin beads despite lacking a chitin binding domain.
    Figure Legend Snippet: Site-directed mutagenesis of conserved cysteines located in the chitin binding domain of P. gallinaceum chitinase ( rPgCHT1 ) and evaluation of chitin binding affinity of mutant rPgCHT1 and rPfCHT1 . Three conserved three cysteines (A) that were selected for mutation using the QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene) with the known template ( PgCHT1 - PET32b plasmid DNA expression construct) containing a thioredoxin epitope tag (TRX-Tag). The changes of single base pairs were achieved within codons 452 aa (1,355 bp, TGC to TCC), 501 aa (1,502 bp, TGC to TCC) and 557 aa (1,670 bp, TGT to TCT) of the plasmid constructs to mutate cysteine (C) residues to serine (S) as shown in (B) . The PfCHT1 - pET-32b expression plasmid was obtained from Gene Universal (Newark DE). The #1 mutant plasmid (in which all three C were modified), #2 plasmid (only two C modified), and wild type (3, rPgCHT2 -PET32) and rPfCHT1 were transformed into SHuffle T7 Express Competent E. coli cells for protein expression. The rPgCHT1 lysates were incubated with chitin beads, washed three times either with either PBS (1, 2, 3) or PBS with 1% Triton X-100 (1-Tx, 2-Tx, and 3-Tx) to remove non-specific or loosely bound chitinase, and evaluated for binding affinity of the mutant rPgCHT1. Similarly, primary E. coli lysate containing rPfCHT1 in soluble form Ni-NTA purified rPfCHT1 was incubated with chitin beads and washed with PBS and PBST (0.2%–1% Triton-X 100). The bound chitin beads were mixed with sample buffer and subjected to SDS-PAGE followed by Western immunoblot analysis with anti-thioredoxin monoclonal antibody (Abcam) to detect rPgCHT1, and 1C3 antibody to detect rPfCHT1 (E, F) . Panel (C) shows the expression of rPgCHT1 (75 kDa) protein in all clones. The binding affinity to chitin beads of mutant #58 (all three cysteines modified) was lower and largely eliminated with the Triton-X 100 wash (D) , indicating that the chitin binding domain is essential for the interaction of the long form chitinase ( PgCHT1 ) to chitin. Panels (E, F) show the strong binding of rPfCHT1 to chitin beads in both (E) coli culture lysates and purified rPfCHT1. In (E, F) , chitin beads were washed with PBS with different concentrations of Triton X 100 (0%–1%). rPfCHT1 strongly binds to chitin beads despite lacking a chitin binding domain.

    Techniques Used: Mutagenesis, Binding Assay, Plasmid Preparation, Expressing, Construct, Positron Emission Tomography, Modification, Transformation Assay, Incubation, Purification, SDS Page, Western Blot, Clone Assay

    7) Product Images from "Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System"

    Article Title: Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2016.00057

    SDS-PAGE (top pictures) and immunodetection (bottom pictures) of C-terminal HFBI-fusion proteins expressed in E. coli Shuffle T7 Express . (A) BMO–HFBI and (B) BMR–HFBI. (1) Whole-cells before induction; (2) cells 1 h post-induction; (3) cells 2 h post-induction; (4) cells 3 h post-induction; (5) cells 4 h post-induction; (6) purified BMO–HFBI or BMR–HFBI of cells harvested 18 h post-induction; (7) purified non-fused BMR or BMO of cells harvested 18 h post-induction. BMO–HFBI (66 kDa) and BMR–HFBI (76 kDa) are marked with a full arrow. Non-fused BMO (57 kDa) and BMR (68 kDa) are marked with an open arrow. Approximately 10 μg total protein per lane. M, molecular weight marker.
    Figure Legend Snippet: SDS-PAGE (top pictures) and immunodetection (bottom pictures) of C-terminal HFBI-fusion proteins expressed in E. coli Shuffle T7 Express . (A) BMO–HFBI and (B) BMR–HFBI. (1) Whole-cells before induction; (2) cells 1 h post-induction; (3) cells 2 h post-induction; (4) cells 3 h post-induction; (5) cells 4 h post-induction; (6) purified BMO–HFBI or BMR–HFBI of cells harvested 18 h post-induction; (7) purified non-fused BMR or BMO of cells harvested 18 h post-induction. BMO–HFBI (66 kDa) and BMR–HFBI (76 kDa) are marked with a full arrow. Non-fused BMO (57 kDa) and BMR (68 kDa) are marked with an open arrow. Approximately 10 μg total protein per lane. M, molecular weight marker.

    Techniques Used: SDS Page, Immunodetection, Purification, Molecular Weight, Marker

    8) Product Images from "Tetrahymena thermophila Granule Lattice Protein 3 Improves Solubility of Sexual Stage Malaria Antigens Expressed in Escherichia coli"

    Article Title: Tetrahymena thermophila Granule Lattice Protein 3 Improves Solubility of Sexual Stage Malaria Antigens Expressed in Escherichia coli

    Journal: bioRxiv

    doi: 10.1101/2021.06.29.450425

    Expression of Pfs25-TEV-Grl1 fusion protein under different conditions. Shuffle T7 Express E. coli cells were induced with increasing concentrations of IPTG at 16°C, harvested and then lysed by sonication 4 hr after induction. Followig high-speed centrifugation, the cell pellet and soluble protein fractions were subjected to SDS-PAGE and either stained with Coomassie blue (A) or processed for Western botting using an antibody against the C-terminal His-tag (B). Note the presence of a prominent band of ∼ 90kDa in the insoluble fractions that stained heavily with anti-His antibody in the Western blot. Positively stained material was also present in the soluble fractions. When applied to Ni-NTA and eluted with increasing concentrations of imidazole (Wash: 10mM, Eluate 1: 50mM, Eluate 2: 100mM. Eluate 3: 200mM), the soluble fractions yielded a highly purified protein of the expected size of the fusion protein on SDS-PAGE (C).
    Figure Legend Snippet: Expression of Pfs25-TEV-Grl1 fusion protein under different conditions. Shuffle T7 Express E. coli cells were induced with increasing concentrations of IPTG at 16°C, harvested and then lysed by sonication 4 hr after induction. Followig high-speed centrifugation, the cell pellet and soluble protein fractions were subjected to SDS-PAGE and either stained with Coomassie blue (A) or processed for Western botting using an antibody against the C-terminal His-tag (B). Note the presence of a prominent band of ∼ 90kDa in the insoluble fractions that stained heavily with anti-His antibody in the Western blot. Positively stained material was also present in the soluble fractions. When applied to Ni-NTA and eluted with increasing concentrations of imidazole (Wash: 10mM, Eluate 1: 50mM, Eluate 2: 100mM. Eluate 3: 200mM), the soluble fractions yielded a highly purified protein of the expected size of the fusion protein on SDS-PAGE (C).

    Techniques Used: Expressing, Sonication, Centrifugation, SDS Page, Staining, Western Blot, Purification

    9) 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

    10) Product Images from "A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate"

    Article Title: A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate

    Journal: Viruses

    doi: 10.3390/v12040385

    SDS-PAGE of the Qm1 expression in fermentation. SDS-PAGE with Coomassie blue staining under reducing conditions after 6 h of induction. MWP: molecular weight pattern; SRF1, SRF2, and SRF3: rupture supernatants from each of the three fermentations of the Qm1 with SHuffle ® T7 Express clone; PRF1, PRF2, and PRF3: the pellet of rupture from each of the three fermentations of Qm1 SHuffle ® T7 Express clone; C-SR and C-PR: the rupture supernatants and pellet of rupture from the fermentation negative control sample. PRC +: the previous sample obtained positive detected by Western blot with an anti-his antibody. We used 20 µL per sample.
    Figure Legend Snippet: SDS-PAGE of the Qm1 expression in fermentation. SDS-PAGE with Coomassie blue staining under reducing conditions after 6 h of induction. MWP: molecular weight pattern; SRF1, SRF2, and SRF3: rupture supernatants from each of the three fermentations of the Qm1 with SHuffle ® T7 Express clone; PRF1, PRF2, and PRF3: the pellet of rupture from each of the three fermentations of Qm1 SHuffle ® T7 Express clone; C-SR and C-PR: the rupture supernatants and pellet of rupture from the fermentation negative control sample. PRC +: the previous sample obtained positive detected by Western blot with an anti-his antibody. We used 20 µL per sample.

    Techniques Used: SDS Page, Expressing, Staining, Molecular Weight, Negative Control, Western Blot

    The expression analysis of Chimera 1 (Qm1) in Escherichia coli (SHuffle ® T7 Express) under denaturant conditions. SDS-PAGE ( A ) and Western blot assay ( B ) using monoclonal anti-histidine antibodies (Clontech). MWP: molecular weight pattern, 1–6: six transformant colonies. 7: untransformed SHuffle ® T7 Express E. coli strain used as a negative control. All samples were processed after 6 h of induction.
    Figure Legend Snippet: The expression analysis of Chimera 1 (Qm1) in Escherichia coli (SHuffle ® T7 Express) under denaturant conditions. SDS-PAGE ( A ) and Western blot assay ( B ) using monoclonal anti-histidine antibodies (Clontech). MWP: molecular weight pattern, 1–6: six transformant colonies. 7: untransformed SHuffle ® T7 Express E. coli strain used as a negative control. All samples were processed after 6 h of induction.

    Techniques Used: Expressing, SDS Page, Western Blot, Molecular Weight, Negative Control

    The identification of Qm1 expressed in the SHuffle ® T7 Express E. coli strain. ( A ) SDS-PAGE Coomassie blue staining in the reducing condition (left) and Western blot (right) using a polyclonal anti-Cap antibody, previously released against proteins of the SHuffle ® T7 Express strain of E. coli . ( B ) The same as ( A ), but SDS-PAGE under non-reducing conditions. MWP: molecular weight pattern; SR: supernatants of cell rupture; PR: pellet of cell rupture of the Qm1 clone 1. C-SR and C-PR correspond to the negative control sample of the SHuffle ® T7 Express. The white arrows indicate protein aggregates detected by the antibody. A volume of 20 µL per sample was used in each case in both conditions.
    Figure Legend Snippet: The identification of Qm1 expressed in the SHuffle ® T7 Express E. coli strain. ( A ) SDS-PAGE Coomassie blue staining in the reducing condition (left) and Western blot (right) using a polyclonal anti-Cap antibody, previously released against proteins of the SHuffle ® T7 Express strain of E. coli . ( B ) The same as ( A ), but SDS-PAGE under non-reducing conditions. MWP: molecular weight pattern; SR: supernatants of cell rupture; PR: pellet of cell rupture of the Qm1 clone 1. C-SR and C-PR correspond to the negative control sample of the SHuffle ® T7 Express. The white arrows indicate protein aggregates detected by the antibody. A volume of 20 µL per sample was used in each case in both conditions.

    Techniques Used: SDS Page, Staining, Western Blot, Molecular Weight, Negative Control

    11) Product Images from "Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm"

    Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm

    Journal: Biotechnology progress

    doi: 10.1002/btpr.3102

    (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value
    Figure Legend Snippet: (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value

    Techniques Used: Western Blot, Expressing, Recombinant, Produced, Bradford Assay, Software, Standard Deviation

    (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.
    Figure Legend Snippet: (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.

    Techniques Used: Western Blot, Expressing, Binding Assay, Activity Assay, Purification, Construct, Standard Deviation

    SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.
    Figure Legend Snippet: SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.

    Techniques Used: SDS Page, Construct, Purification, Molecular Weight, Marker

    12) Product Images from "Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm"

    Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm

    Journal: Biotechnology progress

    doi: 10.1002/btpr.3102

    (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value
    Figure Legend Snippet: (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value

    Techniques Used: Western Blot, Expressing, Recombinant, Produced, Bradford Assay, Software, Standard Deviation

    (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.
    Figure Legend Snippet: (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.

    Techniques Used: Western Blot, Expressing, Binding Assay, Activity Assay, Purification, Construct, Standard Deviation

    SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.
    Figure Legend Snippet: SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.

    Techniques Used: SDS Page, Construct, Purification, Molecular Weight, Marker

    13) Product Images from "Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm"

    Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm

    Journal: Biotechnology progress

    doi: 10.1002/btpr.3102

    (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value
    Figure Legend Snippet: (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value

    Techniques Used: Western Blot, Expressing, Recombinant, Produced, Bradford Assay, Software, Standard Deviation

    (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.
    Figure Legend Snippet: (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.

    Techniques Used: Western Blot, Expressing, Binding Assay, Activity Assay, Purification, Construct, Standard Deviation

    SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.
    Figure Legend Snippet: SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.

    Techniques Used: SDS Page, Construct, Purification, Molecular Weight, Marker

    14) Product Images from "Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli"

    Article Title: Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli

    Journal: bioRxiv

    doi: 10.1101/2020.05.09.085944

    Phenotypic selection of cyclonal variants with differential antigen-binding activity. (a) Representative selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding spTorA-CAT-Gcn4-PP and a second plasmid encoding anti-Gcn4 cyclonal parent (GLF) or variant with CDR-H3 mutation as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM IPTG to induce protein expression. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate. (b) Survival curves for serially diluted SHuffle T7 Express cells co-expressing an anti-Gcn4 cyclonal variant along with the spTorA-CAT-Gcn4-PP reporter. Cells expressing the parental GLF cyclonal along with the non-cognate spTorA-CAT-HAG chimeric antigen (open circle) served as a negative control. Overnight cultures were serially diluted in liquid LB and plated on LB-agar supplemented with Cm. Maximal cell dilution that allowed growth is plotted versus Cm concentration. Arrow in (b) indicates data depicted in image panel (a) and corresponds to 20 μg/ml Cm.
    Figure Legend Snippet: Phenotypic selection of cyclonal variants with differential antigen-binding activity. (a) Representative selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding spTorA-CAT-Gcn4-PP and a second plasmid encoding anti-Gcn4 cyclonal parent (GLF) or variant with CDR-H3 mutation as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM IPTG to induce protein expression. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate. (b) Survival curves for serially diluted SHuffle T7 Express cells co-expressing an anti-Gcn4 cyclonal variant along with the spTorA-CAT-Gcn4-PP reporter. Cells expressing the parental GLF cyclonal along with the non-cognate spTorA-CAT-HAG chimeric antigen (open circle) served as a negative control. Overnight cultures were serially diluted in liquid LB and plated on LB-agar supplemented with Cm. Maximal cell dilution that allowed growth is plotted versus Cm concentration. Arrow in (b) indicates data depicted in image panel (a) and corresponds to 20 μg/ml Cm.

    Techniques Used: Selection, Binding Assay, Activity Assay, Plasmid Preparation, Variant Assay, Mutagenesis, Expressing, Negative Control, Concentration Assay

    Schematic of Tat-dependent positive selection for antigen-binding activity of full-length IgGs. In the absence of a cognate binding protein, a chimeric antigen comprised of a peptide or protein antigen of interest (orange) fused to the C-terminus of spTorA-CAT (blue) is exported out of the cytoplasm by the TatABC translocase. By localizing the chimeric antigen into the periplasm, the CAT domain is no longer able to inactivate the antibiotic chloramphenicol (Cm) by acetylation using acetyl-coenzyme A (CoA) and the host cells are rendered sensitive to antibiotic. When a cyclonal (yellow/black) is functionally expressed in the cytoplasm of SHuffle T7 Express cells, it binds specifically to the chimeric antigen, thereby sequestering the CAT domain in the cytoplasm where it can efficiently detoxify Cm (green star) and conferring an antibiotic-resistant phenotype. Individual clones from the selection plate are selected, genetically identified, and functionally characterized. Yellow balls/sticks represent the 16 intra- and intermolecular disulfide bonds in IgG that are required for folding and activity. (b) Schematic of pBAD24-based vector for expression of chimeric antigen constructs (left) and pCD1-based vector for expression of cyclonal IgGs (right). Abbreviations: RBS, ribosome-binding site; sp, TorA signal peptide; L, flexible GTSAAAG linker; Ag, antigen; V H , variable heavy; V L , variable light; CH, constant heavy; CL, constant light.
    Figure Legend Snippet: Schematic of Tat-dependent positive selection for antigen-binding activity of full-length IgGs. In the absence of a cognate binding protein, a chimeric antigen comprised of a peptide or protein antigen of interest (orange) fused to the C-terminus of spTorA-CAT (blue) is exported out of the cytoplasm by the TatABC translocase. By localizing the chimeric antigen into the periplasm, the CAT domain is no longer able to inactivate the antibiotic chloramphenicol (Cm) by acetylation using acetyl-coenzyme A (CoA) and the host cells are rendered sensitive to antibiotic. When a cyclonal (yellow/black) is functionally expressed in the cytoplasm of SHuffle T7 Express cells, it binds specifically to the chimeric antigen, thereby sequestering the CAT domain in the cytoplasm where it can efficiently detoxify Cm (green star) and conferring an antibiotic-resistant phenotype. Individual clones from the selection plate are selected, genetically identified, and functionally characterized. Yellow balls/sticks represent the 16 intra- and intermolecular disulfide bonds in IgG that are required for folding and activity. (b) Schematic of pBAD24-based vector for expression of chimeric antigen constructs (left) and pCD1-based vector for expression of cyclonal IgGs (right). Abbreviations: RBS, ribosome-binding site; sp, TorA signal peptide; L, flexible GTSAAAG linker; Ag, antigen; V H , variable heavy; V L , variable light; CH, constant heavy; CL, constant light.

    Techniques Used: Selection, Binding Assay, Activity Assay, Clone Assay, Plasmid Preparation, Expressing, Construct

    Genetic selection for cyclonal antigen-binding activity. Selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding one of the chimeric antigens (spTorA-CAT-Ag or an export-defective variant spTorA(KK)-CAT-Ag) alone or with a second plasmid encoding a full-length cyclonal IgG specific for HAG, Gcn4-PP, or c-Myc as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) to induce chimeric antigen and cyclonal expression, respectively. Cross-pairing the anti-HAG cyclonal with non-cognate c-Myc or Gcn4-PP and the anti-Gcn4 cyclonal with non-cognate HAG served as negative controls. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate.
    Figure Legend Snippet: Genetic selection for cyclonal antigen-binding activity. Selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding one of the chimeric antigens (spTorA-CAT-Ag or an export-defective variant spTorA(KK)-CAT-Ag) alone or with a second plasmid encoding a full-length cyclonal IgG specific for HAG, Gcn4-PP, or c-Myc as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) to induce chimeric antigen and cyclonal expression, respectively. Cross-pairing the anti-HAG cyclonal with non-cognate c-Myc or Gcn4-PP and the anti-Gcn4 cyclonal with non-cognate HAG served as negative controls. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate.

    Techniques Used: Selection, Binding Assay, Activity Assay, Plasmid Preparation, Variant Assay, Expressing

    15) Product Images from "Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm"

    Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm

    Journal: Biotechnology progress

    doi: 10.1002/btpr.3102

    (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value
    Figure Legend Snippet: (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value

    Techniques Used: Western Blot, Expressing, Recombinant, Produced, Bradford Assay, Software, Standard Deviation

    (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.
    Figure Legend Snippet: (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.

    Techniques Used: Western Blot, Expressing, Binding Assay, Activity Assay, Purification, Construct, Standard Deviation

    SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.
    Figure Legend Snippet: SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.

    Techniques Used: SDS Page, Construct, Purification, Molecular Weight, Marker

    16) 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

    17) Product Images from "Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm"

    Article Title: Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia colicytoplasm

    Journal: Biotechnology progress

    doi: 10.1002/btpr.3102

    (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value
    Figure Legend Snippet: (a) Soluble/insoluble ratio of anti-HER2 scFv-V L V H expressed in BL21 (DE3) and SHuffle T7 Express, at different induction temperatures (16°C and 30°C). (b) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli SHuffle T7 Express cells expressing anti-HER2 scFv-V L V H . Recombinant proteins were produced at either 30°C (left panel) or 16°C (right panel) and samples were collected 16 h after induction. All samples were normalized by total volume, as determined by the Bradford assay. Relative quantification analysis of Western blots was performed using Image Lab 5.1 software. The band intensities of expressed anti-HER2 scFv-V L V H were divided by the reference band (insoluble fraction) intensity. All data are the average of two independent experiments and error bars are the standard deviation of the mean. ** and *** represent significant difference at P -value

    Techniques Used: Western Blot, Expressing, Recombinant, Produced, Bradford Assay, Software, Standard Deviation

    (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.
    Figure Legend Snippet: (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, T ind =30°C, expressing anti-HER2 scFv-V L V H . Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-V L V H construct expressed in E. coli BL21(DE3) and SHuffle T7 Express cells against HER2 antigen in mammalian cell lysate. The activities measured in PBS alone and BSA in 1x PBS served as negative controls. All data are the average of triplicates and error bars are the standard deviation of the mean.

    Techniques Used: Western Blot, Expressing, Binding Assay, Activity Assay, Purification, Construct, Standard Deviation

    SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.
    Figure Legend Snippet: SDS-PAGE analysis of anti-HER2-scFv constructs expressed in E. coli SHuffle T7 Express at 16°C, 16 h after induction. The cells carrying different constructs were resuspended with equilibration buffer by normalizing Abs 600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-V L V H (lane 2), and pET28a-anti-HER2-scFv-V H V L (lane 4). (b) Purified anti-HER2 scFv-V L V H (lane 5) and anti-HER2 scFv-V H V L (lane 6) constructs. Molecular weight (MW) marker is shown in each gel. The scFv’s were purified with 1 ml Ni-NTA resin, desalted and concentrated with 10 kDa molecular weight cut-off column.

    Techniques Used: SDS Page, Construct, Purification, Molecular Weight, Marker

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    New England Biolabs shuffle t7 express
    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 <t>T7</t> Express was required for soluble expression of some orthologs.
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    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.

    Journal: bioRxiv

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

    doi: 10.1101/2022.02.15.480596

    Figure Lengend 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.

    Article Snippet: For protein expression, BL21 (DE3) Gold, Shuffle T7 express (NEB), Origami B (DE3), Rosetta Gami B (DE3) (EMD-Millipore) competent cells were used.

    Techniques: 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 # ).

    Journal: bioRxiv

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

    doi: 10.1101/2022.02.15.480596

    Figure Lengend 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 # ).

    Article Snippet: For protein expression, BL21 (DE3) Gold, Shuffle T7 express (NEB), Origami B (DE3), Rosetta Gami B (DE3) (EMD-Millipore) competent cells were used.

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

    Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    Article Snippet: To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Techniques: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Staining, Fluorescence, Purification

    Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    Article Snippet: To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Techniques: Expressing, Recombinant, SDS Page, Over Expression

    Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    Article Snippet: To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Techniques: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Western Blot

    Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Large-scale soluble expression of recombinant AaLT-NL zymogen protease grown in TB media at 10 °C (induced with 0.1 mM IPTG). Plasmid construct was transformed into SHuffle® T7 Express Competent E. coli cells (NEB). Samples were collected at the given time points (in hours). The MW ladder is in kilo-Daltons (kDa). Gel analysis of samples collected from the growth of AaLT was first visualized using InVision™ His-Tag In-Gel Stain (Invitrogen), which specifically chelates to and enhances the fluorescence of poly his-tagged proteins (top figure). The His-Tag stain is the positive identification that the bands expressed in the gel below are indeed the expression of soluble AaLT-zymogen (MW ~ 27.6 kDa, red arrows). The growth was extended beyond 24 h due to the 10 °C growth conditions, which helped in solubly expressing the protease, but also to increase bacterial cell density in order to obtain a large quantity of cell paste for purification

    Article Snippet: To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Techniques: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Staining, Fluorescence, Purification

    Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Initial attempt at solubly expressing recombinant midgut proteases in SHuffle® T7 Express Competent E. coli cells (NEB). For each growth experiment, TB media and a 30 °C growth temperature was used. Cells were induced with 0.1 mM IPTG when reaching the log phase (OD 600nm ~ 0.5–1.0). Samples were collected at the given time points (in hours) and prepared for SDS-PAGE analysis. The MW ladder is in kilo-Daltons (kDa). In all cases, the arrow indicates where the expected soluble over-expressed protease should appear. However, all proteases under these conditions were expressed insolubly, only observed in the total samples. a 4–12% BIS-TRIS gel over-expression of AaET grown for a total of 26 h. The MW of the his 6 -tagged AaET-NL zymogen is ~ 27.0 kDa. b 12% BIS-TRIS gel over-expression of AaSPVI grown for a total of 4 h. The MW of the his 6 -tagged AaSPVI-NL zymogen is ~ 28.7 kDa. c 12% BIS-TRIS gel over-expression of AaSPVII grown for a total of 4 h. The MW of the his 6 -tagged AaSPVII-NL zymogen is ~ 28.7 kDa. d 12% BIS-TRIS gel over-expression of AaLT grown for a total of 4 h. The MW of the his 6 -tagged AaLT-NL zymogen is ~ 27.6 kDa

    Article Snippet: To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Techniques: Expressing, Recombinant, SDS Page, Over Expression

    Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    Journal: BMC Biochemistry

    Article Title: Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the Aedes aegypti Mosquito Utilizing E. coli as a host

    doi: 10.1186/s12858-018-0101-0

    Figure Lengend Snippet: Soluble expression of recombinant AaET-NL and AaSPVII-NL zymogen proteases grown in TB media at 23 °C post-induction (induced with 0.1 mM IPTG). Plasmid constructs were transformed into SHuffle® T7 Express Competent E. coli cells (NEB). The MW ladder is in kilo-Daltons (kDa). a Western blot analysis utilizing an AaET-specific antibody of soluble samples collected from the growth and expression of AaET (a total of 4 h post-induction). The zymogen (inactive form of the protease) is observed in the first 2 h (MW ~ 27.0 kDa, red arrow), but a second species hypothesized to be the active mature form begins to appear at the two-hour time-point (MW ~ 22.4 kDa, green arrow) while the zymogen completely disappears by the third hour post-induction. b Large scale expression analysis of AaSPVII-zymogen grown for a total of 5 h post-induction. A single band at ~ 28.7 kDa (orange arrow) is observed to be increasing over time after induction with no observable band present in both the total and soluble pre-induction samples ( t = 0 h)

    Article Snippet: To overcome this, we turned to a BL21(DE3) derivative known as SHuffle® T7 Express Competent E. coli (New England Biolabs #C3029J, Ipswich, MA).

    Techniques: Expressing, Recombinant, Plasmid Preparation, Construct, Transformation Assay, Western Blot

    Phenotypic selection of cyclonal variants with differential antigen-binding activity. (a) Representative selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding spTorA-CAT-Gcn4-PP and a second plasmid encoding anti-Gcn4 cyclonal parent (GLF) or variant with CDR-H3 mutation as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM IPTG to induce protein expression. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate. (b) Survival curves for serially diluted SHuffle T7 Express cells co-expressing an anti-Gcn4 cyclonal variant along with the spTorA-CAT-Gcn4-PP reporter. Cells expressing the parental GLF cyclonal along with the non-cognate spTorA-CAT-HAG chimeric antigen (open circle) served as a negative control. Overnight cultures were serially diluted in liquid LB and plated on LB-agar supplemented with Cm. Maximal cell dilution that allowed growth is plotted versus Cm concentration. Arrow in (b) indicates data depicted in image panel (a) and corresponds to 20 μg/ml Cm.

    Journal: bioRxiv

    Article Title: Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli

    doi: 10.1101/2020.05.09.085944

    Figure Lengend Snippet: Phenotypic selection of cyclonal variants with differential antigen-binding activity. (a) Representative selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding spTorA-CAT-Gcn4-PP and a second plasmid encoding anti-Gcn4 cyclonal parent (GLF) or variant with CDR-H3 mutation as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM IPTG to induce protein expression. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate. (b) Survival curves for serially diluted SHuffle T7 Express cells co-expressing an anti-Gcn4 cyclonal variant along with the spTorA-CAT-Gcn4-PP reporter. Cells expressing the parental GLF cyclonal along with the non-cognate spTorA-CAT-HAG chimeric antigen (open circle) served as a negative control. Overnight cultures were serially diluted in liquid LB and plated on LB-agar supplemented with Cm. Maximal cell dilution that allowed growth is plotted versus Cm concentration. Arrow in (b) indicates data depicted in image panel (a) and corresponds to 20 μg/ml Cm.

    Article Snippet: Bacterial strains E. coli strain DH5α was used for plasmid construction while SHuffle T7 Express (New England Biolabs) was used for cyclonal expression and library selections.

    Techniques: Selection, Binding Assay, Activity Assay, Plasmid Preparation, Variant Assay, Mutagenesis, Expressing, Negative Control, Concentration Assay

    Schematic of Tat-dependent positive selection for antigen-binding activity of full-length IgGs. In the absence of a cognate binding protein, a chimeric antigen comprised of a peptide or protein antigen of interest (orange) fused to the C-terminus of spTorA-CAT (blue) is exported out of the cytoplasm by the TatABC translocase. By localizing the chimeric antigen into the periplasm, the CAT domain is no longer able to inactivate the antibiotic chloramphenicol (Cm) by acetylation using acetyl-coenzyme A (CoA) and the host cells are rendered sensitive to antibiotic. When a cyclonal (yellow/black) is functionally expressed in the cytoplasm of SHuffle T7 Express cells, it binds specifically to the chimeric antigen, thereby sequestering the CAT domain in the cytoplasm where it can efficiently detoxify Cm (green star) and conferring an antibiotic-resistant phenotype. Individual clones from the selection plate are selected, genetically identified, and functionally characterized. Yellow balls/sticks represent the 16 intra- and intermolecular disulfide bonds in IgG that are required for folding and activity. (b) Schematic of pBAD24-based vector for expression of chimeric antigen constructs (left) and pCD1-based vector for expression of cyclonal IgGs (right). Abbreviations: RBS, ribosome-binding site; sp, TorA signal peptide; L, flexible GTSAAAG linker; Ag, antigen; V H , variable heavy; V L , variable light; CH, constant heavy; CL, constant light.

    Journal: bioRxiv

    Article Title: Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli

    doi: 10.1101/2020.05.09.085944

    Figure Lengend Snippet: Schematic of Tat-dependent positive selection for antigen-binding activity of full-length IgGs. In the absence of a cognate binding protein, a chimeric antigen comprised of a peptide or protein antigen of interest (orange) fused to the C-terminus of spTorA-CAT (blue) is exported out of the cytoplasm by the TatABC translocase. By localizing the chimeric antigen into the periplasm, the CAT domain is no longer able to inactivate the antibiotic chloramphenicol (Cm) by acetylation using acetyl-coenzyme A (CoA) and the host cells are rendered sensitive to antibiotic. When a cyclonal (yellow/black) is functionally expressed in the cytoplasm of SHuffle T7 Express cells, it binds specifically to the chimeric antigen, thereby sequestering the CAT domain in the cytoplasm where it can efficiently detoxify Cm (green star) and conferring an antibiotic-resistant phenotype. Individual clones from the selection plate are selected, genetically identified, and functionally characterized. Yellow balls/sticks represent the 16 intra- and intermolecular disulfide bonds in IgG that are required for folding and activity. (b) Schematic of pBAD24-based vector for expression of chimeric antigen constructs (left) and pCD1-based vector for expression of cyclonal IgGs (right). Abbreviations: RBS, ribosome-binding site; sp, TorA signal peptide; L, flexible GTSAAAG linker; Ag, antigen; V H , variable heavy; V L , variable light; CH, constant heavy; CL, constant light.

    Article Snippet: Bacterial strains E. coli strain DH5α was used for plasmid construction while SHuffle T7 Express (New England Biolabs) was used for cyclonal expression and library selections.

    Techniques: Selection, Binding Assay, Activity Assay, Clone Assay, Plasmid Preparation, Expressing, Construct

    Genetic selection for cyclonal antigen-binding activity. Selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding one of the chimeric antigens (spTorA-CAT-Ag or an export-defective variant spTorA(KK)-CAT-Ag) alone or with a second plasmid encoding a full-length cyclonal IgG specific for HAG, Gcn4-PP, or c-Myc as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) to induce chimeric antigen and cyclonal expression, respectively. Cross-pairing the anti-HAG cyclonal with non-cognate c-Myc or Gcn4-PP and the anti-Gcn4 cyclonal with non-cognate HAG served as negative controls. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate.

    Journal: bioRxiv

    Article Title: Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli

    doi: 10.1101/2020.05.09.085944

    Figure Lengend Snippet: Genetic selection for cyclonal antigen-binding activity. Selective spot plating of SHuffle T7 Express cells carrying a plasmid encoding one of the chimeric antigens (spTorA-CAT-Ag or an export-defective variant spTorA(KK)-CAT-Ag) alone or with a second plasmid encoding a full-length cyclonal IgG specific for HAG, Gcn4-PP, or c-Myc as indicated at left. A total of 5 μl of 10-fold serial diluted cells was plated on LB-agar supplemented with 0 or 20 μg/ml chloramphenicol (Cm) as well as 0.4 % arabinose and 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) to induce chimeric antigen and cyclonal expression, respectively. Cross-pairing the anti-HAG cyclonal with non-cognate c-Myc or Gcn4-PP and the anti-Gcn4 cyclonal with non-cognate HAG served as negative controls. Spot plating results are representative of at least three biological replicates. Dashed white lines indicate spot plating data merged from discontinuous region of plate.

    Article Snippet: Bacterial strains E. coli strain DH5α was used for plasmid construction while SHuffle T7 Express (New England Biolabs) was used for cyclonal expression and library selections.

    Techniques: Selection, Binding Assay, Activity Assay, Plasmid Preparation, Variant Assay, Expressing