title: Expression of human milk beta-casein in Escherichia coli: comparison of recombinant protein with native isoforms.
abstract: Studies on physiological function and on structure-function relationships of human milk beta-casein have been limited. In this study, we have introduced the human beta-casein cDNA into vectors designed for expression in Escherichia coli. The inducible T7-based expression system resulted in high-level expression of recombinant beta-casein. The recombinant beta-casein, localized intracellularly in E. coli, was purified to homogeneity and compared with purified native beta-casein, in particular with respect to phosphorylation. The E. coli-produced beta-casein was found to comigrate with the full-length, nonphosphorylated native human beta-casein isoform 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.
authors.affiliation: Symbicom AB, Umeå, Sweden.
authors.forename: L
authors.initials: L
authors.lastname: Hansson
authors.forename: S
authors.initials: S
authors.lastname: Bergström
authors.forename: O
authors.initials: O
authors.lastname: Hernell
authors.forename: B
authors.initials: B
authors.lastname: Lönnerdal
authors.forename: A K
authors.initials: AK
authors.lastname: Nilsson
authors.forename: M
authors.initials: M
authors.lastname: Strömqvist
mesh_terms: D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D002364:Caseins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008895:Milk, Human / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship
chemical_list: D002364:Caseins; D011993:Recombinant Fusion Proteins