Split-enzyme fragment as a single affinity tag that enables protein expression, purification, and functional assays.

Abstract

Expressing, isolating, and characterizing recombinant proteins is crucial to many disciplines within the biological sciences. Different molecular tagging technologies have been developed to enable each individual step of protein production, from expression through purification and characterization. Monitoring the entire production process requires multiple tags or molecular interactions, because no individual tag has provided the comprehensive breadth of utility. An ideal molecular tag is small and does not interrupt expression, solubility, folding or function of the protein being purified and can be used throughout the production process. We adapted and integrated a split-luciferase system (NanoBiT®, Promega ®) to perform the range of techniques essential to protein production. We developed a simple method to monitor protein expression in real time to optimize expression conditions. We constructed a novel affinity chromatography system using the split-luciferase system to enable purification. We adapted western blot analysis, enzyme-linked immunosorbent assay, and cell-based bioassay to characterize the expressed proteins. Our results demonstrate that a single-tag can fulfill all aspects needed throughout protein production.