Thermodynamic parameters for salt‐induced reversible protein precipitation from automated microscale experiments

Abstract

Reversible precipitation can be used as an efficient purification tool for proteins. In addition, identifying conditions under which precipitation or aggregation occurs is of key importance in the bioprocessing and pharmaceutical industry, as this can aid in better formulations and hinder aggregation in chromatography. We have evaluated the precipitation of proteins as determined by light scattering in microplates as a tool for the high-throughput determination of thermodynamic parameters for protein precipitation, with the potential for screening of formulation additives and relevant bioprocess conditions such as pH. This provides a useful complementary technique to existing microplate-based protein thermostability measurements. Using hen egg-white lysozyme and alcohol dehydrogenase as model proteins we have determined the extent of reversible precipitation as a function of ammonium sulfate and sodium chloride concentrations, and also demonstrated global fitting of the data to generate a model where the fraction precipitated can be predicted for any given condition. The global fit provided thermodynamic parameters, including the free energy for protein precipitation, and also allowed an approximate determination of the average size of the structural nucleus that contributes to the free energy of precipitation for each protein. The rapid collection of thermodynamic parameters for protein precipitation, in parallel with protein thermostability measurements, will provide a powerful platform for protein formulation, and also lead to datasets useful for testing theoretical predictions of reversible precipitation based on the molecular modeling of specific protein structure interactions.

Discrimination of soluble and aggregation-prone proteins based on sequence information

“…The top 5 features with highest variable importance include free energies of transfer (WIMW960101), partition coefficient (ZASB820101) of proteins, net charge of protein (x_netcharge and KLEP840101) and isometric point (pI), consistent with previous studies that pI 20, 21 and free energies of transfer 9, 10 are important to classify soluble and aggregation-prone proteins. Partition coefficient is an important parameter related to molecular solubility.…”

Formation of Insoluble Particulates in a Spray-Dried F(ab’)2 Fragment

“…The alternative explanation is dissolution of the insoluble particulates formed during the first 4 weeks’ storage on further storage up to 4 weeks, but this seems unlikely. A reversible precipitation of a protein is known with salt‐induced processes, but thermally induced precipitation is considered to be irreversible . Note that the formation of the insoluble particulates is not evident in the values for k m and k F1 (Table ), which are approximately equal.…”

Assessments on the catalytic and kinetic properties of beta-glucosidase isolated from a highly efficient antagonistic fungus Trichoderma harzianum

“…Total protein and activity of purified β-glucosidase were determined before and after the dialysis using ammonium sulfate precipitation. The purified β-glucosidase was lyophilized and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for further purification and molecular weight determination (AHMAD et al, 2011).…”