title: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, functionally expressed in and purified from Escherichia coli, yeast, and insect cells.
abstract: UDP-GlcNAc 2-epimerase/ManNAc kinase is the key enzyme of sialic acid biosynthesis in mammals. Its functional expression is a prerequisite for early embryogenesis and for the synthesis of several cell recognition motifs in adult organism. This bifunctional enzyme is involved in the development of different diseases like sialuria or hereditary inclusion body myopathy. For a detailed understanding of the enzyme, large amounts of the pure active protein are needed. Different heterologous cell systems were therefore analyzed for the enzyme, which was found to be functionally expressed in Escherichia coli, the yeast strains Saccharomyces cerevisiae and Pichia pastoris, and insect cells. In all these cell types, the expressed enzyme displayed both epimerase and kinase activities. In E. coli, up to 2mg protein/l cell culture was expressed, in yeast cells only 0.4mg/L, while up to 100mg/L, were detected in insect cells. In all three cell systems, insoluble protein aggregates were also observed. Purification from E. coli resulted in 100microg/L pure and structurally intact protein. For insect cells, purification methods were established which resulted in up to 50mg/L pure, soluble, and active protein. In summary, expression and purification of the UDP-GlcNAc 2-epimerase/ManNAc kinase in Sf-900 cells can yield the milligram amounts of protein required for structural characterization of the enzyme. However, the easier expression in E. coli and yeast provides sufficient quantities for enzymatic and kinetic characterization.
authors.affiliation: Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Freie Universität and Humboldt-Universität Berlin, Institut für Biochemie and Molekularbiologie, Berlin-Dahlem, Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Blume
authors.forename: Darius
authors.initials: D
authors.lastname: Ghaderi
authors.forename: Viola
authors.initials: V
authors.lastname: Liebich
authors.forename: Stephan
authors.initials: S
authors.lastname: Hinderlich
authors.forename: Peter
authors.initials: P
authors.lastname: Donner
authors.forename: Werner
authors.initials: W
authors.lastname: Reutter
authors.forename: Lothar
authors.initials: L
authors.lastname: Lucka
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D018411:Spodoptera / Q000235:genetics
chemical_list: D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C482901:UDP-N-acetylglucosamine 2-epimerase - N-acetylmannosamine kinase