Protein Refolding Guided by High-Throughput Differential Scanning Fluorimetry: a case study of an HtrA-Family Bacterial Protease

The high temperature requirement A (HtrA) serine protease family presents an attractive target class with recent interest for antibacterial development. These proteins contain numerous binding sites and regulatory loops and display diverse oligomerization patterns that depend on substrate occupancy and type. HtrA proteins that are purified natively often coelute with peptides and activating species, shifting oligomerization and protein structure to different activated populations. Here, a redesigned approach to HtrA production by purifying the protein from inclusion bodies under denaturing conditions, and screening for optimal refolding buffer composition using biophysical techniques that are function-agnostic and don't rely on any target-specific readouts or assays is described. This systematic workflow will translate to the production of HtrA-family proteins in higher quantities and of purer composition than the current literature standard. ### Competing Interest Statement The authors have declared no competing interest.