Production, Purification, and Characterization of Recombinant β-Lactoglobulin Variants

Due to the increasing global demand for animal proteins, their recombinant production with microorganisms is becoming increasingly important. The whey protein β-lactoglobulin (BLG), characterized by its β-sheet structure, its two highly conserved disulfide bonds, and a free thiol group, is of special interest. Due to its functional properties, it is an important component of many foods and, moreover, it serves as a model protein for structure-function studies in food sciences. Its recombinant production with microorganisms not only enables more sustainable production, but also allows modifications of the BLG structure. After confirming that a wild-type BLG variant (rBLG) recombinantly produced with Escherichia coli and purified possesses the same structural and physicochemical properties as the bovine BLG, rBLG variants without a free thiol group (rBLG-SH), without an outer disulfide bond (rBLG-SS), and without any cysteines (rBLG-C) were produced by targeted mutagenesis. Each recombinant variant was produced and purified to a concentration of up to 400 mg L-1. Removal of the free thiol group in rBLG-SH had minor effects on the globular structure whereas the removal of the outer disulfide bond in rBLG-SS destabilized the protein structure and prevented dimerization while maintaining the globular structure. In contrast, the elimination of all cysteine residues (rBLG-C) prevented the formation of the characteristic β-barrel structure, which significantly altered the functionality of the protein. In general, the variants can be used to better understand the influence of cysteine residues on the structure, behavior and functionality of BLG. In general, the variants may be used to better understand the influence of cysteine residues on the structure, behavior, and functionality of BLG.