The high-temperature requirement A (HtrA) serine protease family presents an attractive target class for antibacterial therapeutics development. These proteins possess dual protease and chaperone functions and contain numerous binding sites and regulatory loops, displaying diverse oligomerization patterns dependent on substrate type and occupancy. HtrA proteins that are natively purified coelute with contaminating peptides and activating species, shifting oligomerization and protein structure to differently activated populations.Here, a redesigned HtrA production results in cleaner preparations with high yields by overexpressing and purifying target protein from inclusion bodies under denaturing conditions, followed by a high-throughput screen for optimal refolding buffer composition using function-agnostic biophysical techniques that do not rely on target-specific measurements. We use Borrelia burgdorferi HtrA to demonstrate the effectiveness of our function-agnostic approach, while characterization with both new and established biophysical methods shows the retention of proteolytic and chaperone activity of the refolded protein. This systematic workflow and toolset will translate to the production of HtrA-family proteins in higher quantities of pure and monodisperse composition than the current literature standard, with applicability to a broad array of protein purification strategies.

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.