Fig. 1

Production of functional recombinant FhCL3 zymogen and FhCL3 propeptide. (a) SDS-PAGE gel showing the activation of the recombinant FhCL3 zymogen. T0: Inactivated recombinant FhCL3 zymogen expressed in the yeast P. pastoris and purified using affinity chromatography. T1 – T5: Activation of the FhCL3 zymogen was initiated by incubating the enzyme in sodium acetate buffer pH 4.5 at 37 °C for 5 h. The progression of activation was observed by removing samples at each hour, adding the inhibitor E-64 before resolving in a 4–12% SDS-PAGE gel. Z, zymogen; MD, mature domain of FhCL3; P, the released propeptide. (b) SDS-PAGE gel showing expression of the recombinant FhCL3 propeptide in E. coli BL21 cells. Lane 1, cell pellet after induction for 3 h at 30 °C; lane 2, supernatant after extraction of the cell pellet; lane 3, wash after recombinant protein binding to the affinity column (Profinia affinity chromatography system); lane 4, the eluted recombinant ppFhCL3. (c) Western blot analysis of recombinant FhCL3 zymogen (1 μg), FhCL3 propeptide (2 μg), and somatic extract of NEJs 24 h (15 μg). Immunoblots were probed with anti-ppFhCL3 polyclonal antibodies raised in rabbit. M, Molecular weight in kDa