Journal: Biotechnology progress

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

doi: 10.1002/btpr.3102

Figure Lengend Snippet: (a) Amino acid sequences of the variable domains of heavy and light chains obtained from Herceptin36 and synthetic linker. (b) Schematic diagram of the pET28a vector containing anti-HER2 scFv antibodies in different orientations.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: Plasmid Preparation

Journal: Biotechnology progress

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

doi: 10.1002/btpr.3102

Figure Lengend Snippet: (a) Western blot analysis of soluble (sol) and insoluble (ins) fractions from E. coli BL21(DE3) and SHuffle T7 Express cells, Tind=30°C, expressing anti-HER2 scFv-VLVH. Samples were normalized by total protein. (b) Binding activity of purified anti-HER2 scFv-VLVH 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.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

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

Journal: Biotechnology progress

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

doi: 10.1002/btpr.3102

Figure Lengend Snippet: (a) Soluble/insoluble ratio of anti-HER2 scFv-VLVH 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-VLVH. 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-VLVH 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<0.01 and P-value<0.001, respectively.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

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

Journal: Biotechnology progress

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

doi: 10.1002/btpr.3102

Figure Lengend 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 Abs600 to 40. (a) Total protein from pET28a (lane 1 and lane 3), pET28a-anti-HER2-scFv-VLVH (lane 2), and pET28a-anti-HER2-scFv-VHVL (lane 4). (b) Purified anti-HER2 scFv-VLVH (lane 5) and anti-HER2 scFv-VHVL (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.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

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

Journal: Biotechnology progress

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

doi: 10.1002/btpr.3102

Figure Lengend Snippet: SEC analysis anti-HER2 scFv constructs in different orientations using a Superdex 75 10/300 GL gel filtration column.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: Construct, Filtration

Journal: Biotechnology progress

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

doi: 10.1002/btpr.3102

Figure Lengend Snippet: Antigen binding of purified anti-HER2 scFv constructs against commercial HER2. Histidine-tagged scFv13-R4 was used as negative control. All data are the average of triplicates and error bars are the standard deviation of the mean.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: Binding Assay, Purification, Construct, Negative Control, Standard Deviation

Journal: Biotechnology progress

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

doi: 10.1002/btpr.3102

Figure Lengend Snippet: Binding affinity analysis of purified (a) anti-HER2 scFv-VLVH and (b) anti-HER2 scFv-VHVL measured by SPR. Commercial HER2 was immobilized on CM5 sensor chip and the response of different concentrations of anti-HER2 scFv constructs (ranging from 0.625–80 nM) was compared with an empty flow cell. Equilibrium binding responses at each anti-HER2 scFv concentration were plotted with binding curve obtained from the Hill slope non-linear regression analysis. The calculated Kd values are given in the table.

Article Snippet: Briefly, the pH of the buffer and concentration of commercial recombinant human HER2 (Sino Biological, China) was adjusted for optimal immobilization.

Techniques: Binding Assay, Affinity Purification, Construct, Concentration Assay