Fig. 2: Optimization of split intein constructs to assemble FL-dystrophin.

a The predicted structure of the Hinge 3 domain with the two split sites marked by the arrows. The three residues at each side of the split sites were labeled in blue and red. b, h, n Western blotting analysis of dystrophin expression in HEK293 cells transfected with or without different versions of Dys-N, M, and C constructs at a molar ratio of 4:2:1. HEK293 cell lysate transfected with a FL-dystrophin construct was used as a positive control (FL Ctrl) and the GAPDH was used as a loading control. c–f, i–l, o–r Densitometry quantification of the FL-dystrophin band intensity (c, i, o), the ratio of unassembled N versus FL (d, j, p), the ratio of unassembled M versus FL (e, k, q) and the ratio of unassembled C versus FL (f, l, r) from three biological repeats per condition. One-way ANOVA with Tukey’s multiple comparisons test for three groups and two-tailed unpaired Student’s t-test for two groups. g, m Diagrams showing the M and C constructs at the split sites with the −1 to −3 and +1 to +3 residues in the dystrophin exteins labeled. The mutated amino acids are labeled in purple (g) and yellow (m). Data were mean ± SEM. Source data are provided as a Source Data file.