Food proteins from yeast-based precision fermentation: Simple purification of recombinant ß-lactoglobulin using polyphosphate.

Proteins produced through precision fermentation are often purified through chromatographic methods. Faster and more cost-effective purification methods are desired for food application. Here, we present a simple method for purification of protein produced from yeast, using ß-lactoglobulin secreted from Pichia pastoris as an example. The food-grade salt hexametaphosphate (HMP) was used to precipitate the protein at acidic pH, while the impurities (extracellular polysaccharides; mainly mannan) remained soluble. After re-solubilization of the protein-HMP complex by neutralization, excess HMP was selectively precipitated using calcium chloride. The protein content of the crude sample increased from 26 to 72 wt% (comparable to purification with anion exchange chromatography), containing only residual extracellular polysaccharides (9 wt%) and HMP (1 wt%). The established method had no significant impact on the structural and functional properties (i.e., ability to form emulsions) of the protein. The presented method shows potential for cost-effective purification of recombinant proteins produced through yeast-based expression systems.

Determination of specific and non-specific protein-protein interactions for beta-lactoglobulin by analytical ultracentrifugation and membrane osmometry experiments.

Protein-protein interactions are essential for the understanding of biological processes. Specific protein aggregation is an important aspect for many biological systems. In particular, electrostatic interactions play the key role for protein-protein interactions, as many amino acids have pH-dependent charge states. Moreover, protein dissociation is directly related to the solution pH, ionic strength, temperature and protein concentration. The subtle interplay between different specific and non-specific interactions is demonstrated for beta-lactoglobulin (BLG) with a focus on low salt concentrations, thus mimicking technically relevant processing conditions. BLG is a well-characterized model system, proven to attain its monomer-dimer equilibrium strongly dependent upon the pH of the solution. In this manuscript, we present a unique combination of analytical ultracentrifugation and membrane osmometry experiments, which quantifies specific and non-specific interactions, i.e. in terms of the dimer dissociation constants and the second osmotic virial coefficient, at pH 3 and 7 and sodium chloride concentrations of 10 mM and 100 mM. This provides direct insight to protein-protein interactions for a system with a concentration-dependent monomer-dimer equilibrium. Moreover, using a coarse-grained extended DLVO model in combination with molecular dynamics simulations, we quantify non-specific monomer-monomer, monomer-dimer and dimer-dimer interactions as well as the binding free energy of BLG dimerization from theoretical calculations. The experimentally determined interactions are shown to be mainly governed by electrostatic interactions and further agree with free energy calculations. Our experimental protocol aims to determine non-specific and specific interactions for a dynamically interacting system and provides an understanding of protein-protein interactions for BLG at low salt concentrations.