Protein formulation through automated screening of pH and buffer conditions, using the Robotein® high throughput facility.
Eur Biophys J
; 50(3-4): 473-490, 2021 May.
Article
em En
| MEDLINE
| ID: mdl-33611612
ABSTRACT
Among various factors, the direct environment (e.g. pH, buffer components, salts, additives, etc.
) is known to have a crucial effect on both the stability and activity of proteins. In particular, proper buffer and pH conditions can improve their stability and function significantly during purification, storage and handling, which is highly relevant for both academic and industrial applications. It can also promote data reproducibility, support the interpretation of experimental results and, finally, contribute to our general understanding of the biophysical properties of proteins. In this study, we have developed a high throughput screen of 158 different buffers/pH conditions in which we evaluated (i) the protein stability, using differential scanning fluorimetry and (ii) the protein function, using either enzymatic assays or binding activity measurements, both in an automated manner. The modular setup of the screen allows for easy implementation of other characterization methods and parameters, as well as additional test conditions. The buffer/pH screen was validated with five different proteins used as models, i.e. two active-site serine ß-lactamases, two metallo-ß-lactamases (one of which is only active as a tetramer) and a single-domain dromedary antibody fragment (VHH or nanobody). The formulation screen allowed automated and fast determination of optimum buffer and pH profiles for the tested proteins. Besides the determination of the optimum buffer and pH, the collection of pH profiles of many different proteins may also allow to delineate general concepts to understand and predict the relationship between pH and protein properties.
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Base de dados:
MEDLINE
Assunto principal:
Beta-Lactamases
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article