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