Preparation of Polyclonal Antibodies to Barley Granule-Bound Amylopectin Synthase Ia and Their Application in the Characterization of Interacting Proteins

Abstract

The production of amylose is facilitated by granule-bound starch synthase (GBSS). Despite its importance, the specific protein interactions involving barley grain-bound starch synthase Ia (HvGBSSIa) remain poorly understood. To elucidate this, we engineered a pET-32a-HvGBSSIa prokaryotic expression vector for specific expression in E. coli Rosetta cells. A rabbit anti-HvGBSSIa polyclonal antibody was generated and employed to enrich HvGBSSIa-binding proteins from barley grains through immunoprecipitation. The isolated complexes were then resolved through SDS-PAGE, and the constituent proteins were identified using mass spectrometry coupled with database searches. Our results confirmed the successful preparation of a highly specific polyclonal antibody against HvGBSSI. Furthermore, differential expression of HvGBSSIa was assessed across various barley tissues and developmental stages of the grain, revealing peak expression at 25 days post-flowering. Proteins interacting with HvGBSSIa, including sucrose synthase and starch branching enzyme, were identified through co-immunoprecipitation. This study lays the groundwork for further detailed analyses of the HvGBSSIa protein complex in barley.