Fig. 3: Increased stability and folding rate of STAND-A36. | Nature Communications

Fig. 3: Increased stability and folding rate of STAND-A36.

From: An ultra-stable cytoplasmic antibody engineered for in vivo applications

Fig. 3

a Schematic structure of scFvs used in intra-disulfide bond formation analysis (upper: scFv-T7-A36, lower: endoplasmic reticulum (ER)-targeted scFv-A36 (ER-scFv-A36). b Co-localisation of ER-scFv-A36 and ER-RFP in HeLa cells. Arrowheads indicate ER-scFv-A36 in the ER. c Expression of scFv-T7-A36 leads to the formation of cytoplasmic aggregates (arrows). Scale bar, 10 µm. df Western blot analysis of the cell lysate of HeLa cells transfected with vectors expressing scFv-T7-A36 d, ER-scFv-A36 d, STAND-A36 e, and scFv-GFPA36 f under reduced (6% 2-ME or 10 mM DTT) or non-reduced (2-ME, DTT-free) conditions. Differences in antibody migration distances in SDS-PAGE in reduced and non-reduced conditions were observed only in ER-scFv-A36. g SDS–PAGE analysis of the purified STAND-A36 (left), scFv-GFPA36 (middle), and scFv-T7-A36 (right) from E. coli cells under reduced (6% 2-ME or 10 mM DTT) or non-reduced (2-ME, DTT-free) conditions. No differences in migration distance were observed in any of the scFvs (arrowheads). h Analysis of thermal stability of purified STAND-A36, scFv-GFPA36, and scFv-T7-A36 using a fluorescence dye, PSA. The half-lives (t1/2) of the cytoplasmic antibodies at 80 °C were examined (n = 3–4 independent samples). Statistical analysis: two-tailed one-way analysis of variance (t1/2, F (2, 8) = 15.1284, P= 0.001912, Tukey’s multiple comparison test, STAND-A36 vs. scFv-GFPA36: P = 0.001816, STAND-A36 vs. scFv-T7-A36: P = 0.01465, STAND-A36 vs. scFv-T7-A36: P = 0.47826). i Binding affinity of purified STAND-A36, scFv-GFPA36, and scFv-T7-A36 to Syt I-C2A (n = 3 independent samples per group). Scale bars, 10 μm. Error bars represent standard error of the mean. Source data are provided as a Source Data file.

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