Application of biophysical methods for improved protein production and characterization: A case study on an high‐temperature requirement A‐family bacterial protease

Abstract

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.

Protein arginine N-methyltransferase 2 plays a noncatalytic role in the histone methylation activity of PRMT1

“…Proteins displaying multiphasic melting properties in DSF may be indicative of impurities, independently-folded domains, or oligomeric populations with distinct thermal stabilities ( 37 , 49 , 50 ). In this study, we found that human PRMT2 provided a largely biphasic melt depending on protein concentrations.…”

Application of temperature-responsive HIS-tag fluorophores to differential scanning fluorimetry screening of small molecule libraries

“…Borrelia burgdorferi HtrA wildtype (WT) and catalytically-inactive S226A mutant (S/A) were expressed and purified as previously described ( Ronzetti et al, 2022 ). The HIS-tag fluorophores RED-tris-NTA 2 nd Gen (NanoTemper, #MO-L018) and Atto-647 (Sigma, #02175), were both suspended in PBS at 5 μM, aliquoted, and stored at −20°C.…”

“…Beyond screening, there is a significant diversity of manners in which the DSF assay is applied, including buffer and crystallization formulation and binding mechanism studies. Extending these applications, DSF was recently applied to improving refolding conditions of protein after denaturing purifications ( Biter et al, 2016 ; Wang et al, 2017 ; Lee et al, 2019 ; Ronzetti et al, 2022 ). The thermal shift technique has also been applied to more complex protein-protein interactions and for the quantification of overexpressed protein in lysates ( Seo et al, 2014 ; Shao et al, 2020 ).…”