Fig 1. Determining the minimal functional NpuDnaEC intein with PTS activity in mammalian cells.
(A) Protein sequence of systematic truncations of the first N-terminus β-strand of NpuDnaEC intein. (B) Schematic diagram of constructs used for establishing PTS activity, where Lyn is a plasma membrane (PM) localization sequence (shown in purple). Successful PTS activity would result in Cerulean and Venus localized to the PM while mRFP would be localized to the cytoplasm. HeLa cells expressing M-Cerulean-InN-mRFP resulted in (C) CFP and (D) RFP fluorescence localized to the PM. (D) Expression of InC-Venus resulted in YFP fluorescence distributed in the cytoplasm. Co-expression of the two constructs triggered PTS activity resulting PM localization of (F) CFP and (H) YFP while the cytoplasm is (G) RFP fluorescence. Co-expression of M-Cerulean-InN-mRFP with (I-K) t4 and (L-N) t9 truncations of N-terminus of InC in HeLa cells shown in CFP, RFP and YFP fluorescence, respectively. (O) The percentage of co-transfected cells with truncated mutants of InC (n = 6 experiments with over 20 cells). β-strands are shown in orange arrow. Comparison between wild-type: *p < 0.005, ** p < 0.001. Error bars are standard deviation. Scale bars are 10 μm. Images are in false colour.