Abstract
High throughput screening for measuring the stability of industrially relevant proteins and their variants is necessary for quality assessment in the development process. Advances in automation, measurement time and sample consumption for many techniques allow rapid measurements with minimal amount of protein. However, many methods include automated data analysis, potentially neglecting important aspects of the protein's behavior in certain conditions. In this study we implement small angle X-ray scattering (SAXS), typically not used to assess protein behavior in industrial screening, in a high throughput screening workflow to address problems of contradicting results and reproducibility among different high throughput methods. As a case study we use the lipases of Thermomyces lanuginosus and Rhizomucor miehei, widely used industrial biocatalysts. We show that even the initial analysis of the SAXS data without performing any time-consuming modelling provide valuable information on interparticle interactions. We conclude that recent advances in automation and data processing, have enabled SAXS to be used more widely as a tool to gain in-depth knowledge highly useful for protein formulation development. This is especially relevant in light of increasing accessibility to SAXS due to the commercial availability of benchtop instruments.
Highlights
In most cases, the development of industrially relevant proteins in volves many different variants of a naturally occurring protein, pro duced by protein engineering
When measured over the whole pH range, Nano differential scanning fluorimetry (nanoDSF) and isothermal chemical dena turation (ICD) showed that the conformational stability of Thermomyces lanuginosus lipase (TLL) was highest at pH 6 (Fig. S3)
Our case study of the industrial relevant lipases from Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) showed how small angle X-ray scattering (SAXS) gives valuable information on the solution behavior of a protein when included into a systematic formulation screen
Summary
The development of industrially relevant proteins in volves many different variants of a naturally occurring protein, pro duced by protein engineering. Production of industrially relevant proteins, such as enzymes, typically follows an established workflow of different phases [1,2]; high throughput generation of mutants, followed by activity screening. For enzymes or proteins that show desired properties, more in-depth char acterization is carried out This involves determining stability, which is typically done by a range of high throughput and automated biophysical methods. While these techniques are rapid and consume low amounts of sample, information about protein–protein interaction and structural knowledge is severely limited. Small angle X-ray scattering is not commonly used as a high throughput or screening method, but is a powerful tool for structural determination of proteins in solution at low Abbreviations: C, degree Celsius; DLS, dynamic light scattering; GnHCl, Guanidine hydrochloride; ICD, isothermal chemical denaturation; kD, Diffusion interaction parameter; kDa, kilo dalton; MW, Molecular weight; nanoDSF, Nano differential scanning fluorimetry; NMR, Nuclear magnetic resonance; P(r), distance distribution (function); Rg, Radius of gyration; Rh, Hydrodynamic radius; RML, Rhizomucor miehei lipase; Dmax, maximum particle dimension; SAXS, Small-angle X-ray scattering; TLL, Thermomyces lanuginosus lipase
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