shuffle t7 express  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97

    Structured Review

    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 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/shuffle t7 express/product/New England Biolabs
    Average 97 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    shuffle t7 express - by Bioz Stars, 2022-08
    97/100 stars

    Images

    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 "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

    3) 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

    4) 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

    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 "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

    7) 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

    8) 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

    9) 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

    10) 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

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    New England Biolabs shuffle strain c3030
    Cytoplamic expression of proTHI-TRX fusion proteins in strain <t>C3030.</t> For all fusion proteins, 1 ml of cell culture was pelleted and dissolved in 100 μl sample buffer. 10 μl from this extract and an equivalent amount for total soluble cytoplasmic fractions were separated on Tricine/SDS gels. (M) Protein marker, ( 1 ) un-induced crude extract, ( 2 ) induced crude extract, ( 3 ) total soluble fraction taken after cell lysis by sonication, ( 4 ) insoluble fraction after sonication. Red stars indicate the 25 kDa fusion protein
    Shuffle Strain C3030, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/shuffle strain c3030/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    shuffle strain c3030 - by Bioz Stars, 2022-08
    93/100 stars
      Buy from Supplier

    96
    New England Biolabs e coli t7 shuffle cells
    Comparison of recombinant expression yields in <t>E.</t> <t>coli</t> and insect <t>cells.</t> Expression in S2 cells results in higher yields, purity and shows no correlation with construct length. ( A ) Size exclusion chromatograms and SDS-gel (inset) of shortest (dashed line) and longest (full line) ISG65 constructs expressed in <t>E.</t> <t>coli</t> and insect cells. Bacterial culture volumes were tenfold higher. The protein gel was digitally cropped for clarity. The complete gel is shown in Supplementary Figure 3 . ( B ) Expression yields normalised to 1 L of cell culture. Normalised yields were 38-fold (longest construct, dark grey), and, respectively, 250-fold higher (shortest construct, light grey) for the same constructs expressed in insect cells. EC, E. coli ; DM Drosophila melanogaster; M, Marker.
    E Coli T7 Shuffle Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli t7 shuffle cells/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e coli t7 shuffle cells - by Bioz Stars, 2022-08
    96/100 stars
      Buy from Supplier

    97
    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
    https://www.bioz.com/result/shuffle t7 express/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    shuffle t7 express - by Bioz Stars, 2022-08
    97/100 stars
      Buy from Supplier

    Image Search Results


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

    Journal: Biotechnology Letters

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

    doi: 10.1007/s10529-013-1180-z

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

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

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

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

    Journal: Biotechnology Letters

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

    doi: 10.1007/s10529-013-1180-z

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

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

    Techniques: Purification, Staining, Western Blot, Marker

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-022-16958-x

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

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

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-022-16958-x

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

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

    Techniques: Construct, SDS Page, Molecular Weight, Marker

    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

    The domain structure and catalytic mechanism of AppA. ( a ) A cartoon representation of the structure of Escherichia coli periplasmic phytase (AppA) (α-helices shown as cylinders and β-sheet as arrows). AppA folds to provide an α-domain (upper) and an α/β-domain (lower). The active site cleft (region indicated in pink) is found at their interface. ( b ) Steps in the catalytic mechanism. Core active site residues including selected residues of the nucleophilic (RHGxR, residues 16–20) and proton donor (HDT, residues 303-305) motifs are shown. (1) Nucleophilic attack by H17 to form phosphohistidine intermediate. D304 acts as general acid leading to release of the lower phosphorylated inositol; (2) D304 catalyses breakdown of the phosphohistidine intermediate by acting as a general base; and (3) orthophosphate-bound state. Note that regeneration of the enzyme can occur by donation of the inorganic phosphate group to acceptors other than water.

    Journal: International Journal of Molecular Sciences

    Article Title: Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA

    doi: 10.3390/ijms23116346

    Figure Lengend Snippet: The domain structure and catalytic mechanism of AppA. ( a ) A cartoon representation of the structure of Escherichia coli periplasmic phytase (AppA) (α-helices shown as cylinders and β-sheet as arrows). AppA folds to provide an α-domain (upper) and an α/β-domain (lower). The active site cleft (region indicated in pink) is found at their interface. ( b ) Steps in the catalytic mechanism. Core active site residues including selected residues of the nucleophilic (RHGxR, residues 16–20) and proton donor (HDT, residues 303-305) motifs are shown. (1) Nucleophilic attack by H17 to form phosphohistidine intermediate. D304 acts as general acid leading to release of the lower phosphorylated inositol; (2) D304 catalyses breakdown of the phosphohistidine intermediate by acting as a general base; and (3) orthophosphate-bound state. Note that regeneration of the enzyme can occur by donation of the inorganic phosphate group to acceptors other than water.

    Article Snippet: Expression of 6H3C-AppA and its variants was performed using the E. coli strain Shuffle Express T7 (New England Biolabs).

    Techniques: