Studies on physiological function and on structure-function relationships of human milk β-casein have been limited. In this study, we have introduced the human β-casein cDNA into vectors designed for expression in Escherichia coli. The inducible T7-based expression system resulted in high-level expression of recombinant β-casein. The recombinant β-casein, localized intracellularly in E. coli, was purified to homogeneity and compared with purified native β-casein, in particular with respect to phosphorylation. The E. coli-produced β-casein was found to comigrate with the full-length, nonphosphorylated native human β-casein iso-form on SDS-PAGE. An N-terminal peptide containing all tentative phosphorylation sites was isolated from the recombinant protein and analyzed by mass spectrometry. The molecular mass as well as the migration of this peptide on reversed-phase chromatography confirmed that it was unphosphorylated.
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