Application of biophysical methods for improved protein production and characterization: a case study on an HtrA-family bacterial protease

Abstract

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.

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

“…Borrelia burgdorferi HtrA wildtype (WT) and catalyticallyinactive 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).…”