Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: SDS-PAGE and Western blot analysis of hScFv-IL-17A proteins expressed in various E. coli strains. (a) SDS-PAGE of insoluble fractions; (b) SDS-PAGE of soluble fractions; (c) Western blot of insoluble fractions; (d) Western blot of soluble fractions. Lane 1: M (molecular weight marker); Lanes 2–4: hScFv-IL-17A-WT expressed in E. coli Origami B (DE3), SHuffle, and BL21 (DE3) with DisCoTune, respectively; Lanes 5–7: hScFv-IL-17A-C97S expressed in the same strains; Lane 8: purified hScFv M6-1B9 (positive control). Western blot detection was performed using an anti-His tag antibody. The arrow indicates the expected size of hScFv-IL-17A (∼28 kDa). Estimated amounts of hScFv-IL-17A are labeled. < LOD indicates signals below the limit of detection.

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: SDS Page, Western Blot, Molecular Weight, Marker, Purification, Positive Control, Labeling

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: The expression efficiency of soluble hScFv-IL-17A-WT (orange) and hScFv-IL-17A-C97S (green) in different expression systems: (WT/Ori and C97S/Ori) from Origami B (DE3); (WT/SHuffle and C97S/SHuffle) from SHuffle; and (WT/BL21Dc and C97S/BL21Dc) from BL21 (DE3) with DisCoTune. Data represent mean ± SD from three independent biological replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Expressing

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Indirect ELISA of hScFv-IL-17A binding to hIL-17A using an avidin–biotin system. Purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S were tested at 1 and 5 µg/mL, expressed from SHuffle (WT/SHuffle-1, WT/SHuffle-5; C97S/SHuffle-1, C97S/SHuffle-5; light/dark green) and BL21 (DE3) with DisCoTune (WT/BL21Dc-1, WT/BL21Dc-5; C97S/BL21Dc-1, C97S/BL21Dc-5; light/dark orange). An IL-17A mAb (dark grey) was used as a positive control. Data represent mean ± SD from three independent biological replicates. Statistical analysis was determined by unpaired two-tailed Student’s t -test. Significance levels are denoted as: ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Indirect ELISA, Binding Assay, Avidin-Biotin Assay, Purification, Positive Control, Two Tailed Test

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Binding assessment of hScFv-IL-17A-WT (a) and hScFv-IL-17A-C97S (b) from BL21 (DE3) with DisCoTune by competitive ELISA. Various concentrations (0.1, 1, and 2 µg/mL) of IL-17A mAb (neutralizing antibody) were tested in competition with hScFv-IL-17A. WT was used at 1 µg/mL, whereas C97S was used at 5 µg/mL to compensate for its reduced binding activity observed in indirect ELISA. An anti-IFN-γ mAb (clone B27) was used as an irrelevant antibody control. Data represent mean ± SD from three independent biological replicates.

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Binding Assay, Competitive ELISA, Activity Assay, Indirect ELISA, Control

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Structural alignment of hScFv-IL-17A-WT and hScFvIL-17A-C97S. The predicted structures were generated using ABodyBuilder2. The conformational structures of hScFv-IL-17A-WT and hScFv-IL-17A-C97S were compared using UCSF ChimeraX software. The red ribbon indicates hScFv-IL-17A-WT structure, and the gold ribbon indicates hScFv-IL-17A-C97S structure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Generated, Software