Fig. 5: OsISA2 possesses stronger amylopectin-binding activity and promotes the amylolytic activity of OsISA1.

a Schematic domain of OsISA1, OsISA2 and related protein constructs. OsISA1 and related truncations or mutations are fused with a N-terminal 6×His and a C-terminal myc-tag. OsISA2 and related truncations are constructed with a N-terminal Sumo and a C-terminal Strep-tag. Amylopectin-binding activity of OsISA1 (b) and OsISA2 (c) analyzed by SDS-PAGE and Western blot validation. Bands are categorized as input (In), supernatant (S), wash (W), and pellet (P). The amylopectin-bound ratio (P/In) for each protein is calculated and shown below the gel, with band intensities estimated using ImageJ. 30 nM protein and 60 mg amylopectin were used in each assay. Asterisks indicate the position of the target protein. d OsISA2 enhances OsISA1’s debranching activity as analyzed by native PAGE containing amylopectin. Increasing amounts of OsISA2 result in sharper retardation and more pronounced smearing bands by the formation of OsISA1-ISA2 hetero-multimers. Equal amounts of OsISA1 (50 nM) were used in lanes 2–5. At least three biological replicates were performed.