Fig. 4.

Charge engineering of the NpuDnaE intein. (A) Charge distributions of the NpuDnaE and the Charge-Swapped NpuDnaE (CS-NpuDnaE) inteins corresponding to strands β3 and β6 in the gp41–1 intein structure. Color-coded arrows indicate the pairs of split inteins tested for trans-splicing in panel C with lines. (B) Cis-splicing analysis of the CS-NpuDnaE intein by SDS-PAGE. M, 0h, 3h, and E stand for molecular markers, 0 hours, 3 hours after induction, and elution from Ni-NTA spin columns. A red arrow indicates the band corresponding to the cis-spliced H₆-GB1-GB1 product. A repesentative gel of three individual experiments is shown. (C) Cross-activity between split NpuDnaE and CS-NpuDnaE inteins. SDS-PAGE analysis of trans-splicing for three different pairs of split inteins, CS-NpuDnaE_N/CS-NpuDnaE_C (left), CS-NpuDnaE_N/NpuDnaE_C (middle), and NpuDnaE_N/CS-NpuDnaE_C (right), which are illustrated by arrows and lines in panel A. N, L, and C denote the N-terminal fragment with Int_N, the ligated product, and the C-terminal fragment with Int_C, respectively. I, A, I+A, and E stand for IPTG induction, arabinose induction, both IPTG and arabinose induction, and elution from Ni-NTA spin columns. IPTG induction produces the N-terminal precursor fragment, N. Arabinose induces the protein expression of the C-terminal precursor, C. Only dual induction by IPTG and arabinose (I+A) is expected to produce the ligated product, L by protein trans-splicing. A repesentative gel of two individual experiments for each combination is shown.