Journal: Biotechnology Reports

Article Title: Comparative analysis of anti-MICA scFv affinities: Insights from three label-free biophysical methods and biological validation

doi: 10.1016/j.btre.2026.e00955

Figure Lengend Snippet: Characterization of Recombinant Proteins: MICA and anti-MICA scFvs. (A) Molecular model, shown as a ribbon representation, of the variable fragment of the anti-MICA scFvs. The framework is displayed in white, the light chain CDRs are shown in cyan, and the heavy chain CDRs are shown in yellow. The residues with mutations are shown as magenta spheres [residues 32 (CDR L1), 164 (CDR H1), and 188/190 (CDR H2)]. (B) Schematic diagram of the scFv gene. The modified pET-15b vector was used for the expression of the WT and Beta mutant scFvs, each carrying four mutations: I32Y in CDR1 of the VL, and S164F, P188W, and G190W in CDR1, CDR2, and CDR2 of the VH, respectively. Recombinant proteins were expressed in E. coli BL21(DE3). (C) SDS-PAGE analysis showing the purity of recombinant proteins: WT scFv, Beta mutant scFv, and MICA. Proteins were resolved on a 12% acrylamide gel under reducing conditions. SDS-PAGE results show the soluble fraction (SF), unbound protein (UBP), elution of purified scFv (E), renatured proteins (R) and inclusion bodies (IB). MW, molecular weight. (D-E) Western blot analysis confirming the identity of scFvs and MICA using an anti-HisTag antibody. For the identification of the WT and Beta mutant scFvs, Anti-6xHis Epitope Tag mouse monoclonal antibody conjugated with peroxidase (200-303-382) was used at a dilution of 1:1000. For the identification of MICA, a biotinylated Anti-MICA antibody (BAMO3 (BAFI300, BamOmaB)) and Streptavidin were used at a dilution of 1:2000. A total of 2 μg of purified protein was loaded. The negative control (Ctrl -) for MICA detection was WT scFv and MICA protein was used for scFv detection. Original gel is presented in Fig. S1, Supplementary information.

Article Snippet: The identity of MICA and scFvs proteins was confirmed by western blot using a HRP-conjugated anti-His tag monoclonal antibody (200-303-382, Rockland, USA).

Techniques: Recombinant, Modification, Plasmid Preparation, Expressing, Mutagenesis, SDS Page, Acrylamide Gel Assay, Purification, Molecular Weight, Western Blot, Negative Control

Journal: Research in Pharmaceutical Sciences

Article Title: Enhancing soluble expression and purification of a bispecific scFv antibody in Escherichia coli using chaperones

doi: 10.4103/RPS.RPS_10_24

Figure Lengend Snippet: (A) Schematic presentation of the pET28a-scFv-BsAb expression vector; (B) SDS-PAGE and western blot analysis of bacterial expression of histidine-tagged scFv-BsAb with or without chaperones. Lane 1: without chaperone before induction; lanes 2-4: without chaperone after induction by IPTG (0.25, 0.5, and 1 mM, respectively); lanes 5, 7, and 9: co-expression with chaperones before induction; lanes 6, 8, and 10: co-expression with chaperones after induction by IPTG (0.25, 0.5, and 1 mM, respectively); lane M: Thermofisher protein molecular weight marker 26610; Lane 11: western blot analysis using anti-His-HRP antibody. scFv, Single-chain variable fragment; IPTG, isopropyl-β-D-1-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Article Snippet: To confirm the protein expression and compare the soluble expression level in each condition, the samples were loaded on 12% sodium dodecyl sulfate-polyacrylamide gels, and electrophoresis was performed (SDS-PAGE) with a constant voltage of 150 V. Also, to confirm the expression of the His-tagged scFv-BsAb, the whole protein sample, which was first analyzed by SDS-PAGE, was transferred to polyvinylidene difluoride (PVDF) membranes, and western blotting was done using anti-His (C-term)-horseradish peroxidase (HRP) antibody (Invitrogen, USA).

Techniques: Expressing, Plasmid Preparation, SDS Page, Western Blot, Molecular Weight, Marker, Polyacrylamide Gel Electrophoresis

Journal: Research in Pharmaceutical Sciences

Article Title: Enhancing soluble expression and purification of a bispecific scFv antibody in Escherichia coli using chaperones

doi: 10.4103/RPS.RPS_10_24

Figure Lengend Snippet: Fluorescence intensity histogram based on the results of flow cytometry of PBMCs and HEK293 cells after incubation with scFv-BsAb and a secondary antibody (anti-His-FITC). The lymphocyte population in PBMCs was gated based on forward scatter and side scatter. scFv, Single-chain variable fragment.

Article Snippet: To confirm the protein expression and compare the soluble expression level in each condition, the samples were loaded on 12% sodium dodecyl sulfate-polyacrylamide gels, and electrophoresis was performed (SDS-PAGE) with a constant voltage of 150 V. Also, to confirm the expression of the His-tagged scFv-BsAb, the whole protein sample, which was first analyzed by SDS-PAGE, was transferred to polyvinylidene difluoride (PVDF) membranes, and western blotting was done using anti-His (C-term)-horseradish peroxidase (HRP) antibody (Invitrogen, USA).

Techniques: Fluorescence, Flow Cytometry, Incubation