Production of phosphorylated and functional αs1-casein in Escherichia coli

The study demonstrates two strategies, phosphorylation of αs1-casein using Bacillus subtilis kinases and phosphomimetic substitution of αs1-casein, to generate functional recombinant αs1-casein in Escherichia coli. Mass spectrometry and two-dimensional gel electrophoresis (2DE) confirm site-specific phosphorylation, while functional assays reveal calcium-binding capacity and structural mimicry, offering a foundation for animal-free dairy protein innovation.