title: Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites.
abstract: The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy.
authors.affiliation: Department of Organic Chemistry, University of Konstanz, D-78464 Konstanz, Germany. marina.rubini@uni-konstanz.de.
authors.forename: E
authors.initials: E
authors.lastname: Hoffmann
authors.affiliation: Leibniz Institute of Molecular Pharmacology, D-13125 Berlin and Humboldt Universität zu Berlin, D-12489 Berlin, Germany. hackenbe@fmp-berlin.de.
authors.forename: K
authors.initials: K
authors.lastname: Streichert
authors.affiliation: Leibniz Institute of Molecular Pharmacology, D-13125 Berlin and Humboldt Universität zu Berlin, D-12489 Berlin, Germany. hackenbe@fmp-berlin.de.
authors.forename: N
authors.initials: N
authors.lastname: Nischan
authors.affiliation: Department of Biochemical Pharmacology, University of Konstanz, D-78464 Konstanz, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Seitz
authors.affiliation: Department of Biochemical Pharmacology, University of Konstanz, D-78464 Konstanz, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Brunner
authors.affiliation: Leibniz Institute of Molecular Pharmacology, D-13125 Berlin and Humboldt Universität zu Berlin, D-12489 Berlin, Germany. hackenbe@fmp-berlin.de.
authors.forename: S
authors.initials: S
authors.lastname: Schwagerus
authors.affiliation: Leibniz Institute of Molecular Pharmacology, D-13125 Berlin and Humboldt Universität zu Berlin, D-12489 Berlin, Germany. hackenbe@fmp-berlin.de.
authors.forename: C P R
authors.initials: CP
authors.lastname: Hackenberger
authors.affiliation: Department of Organic Chemistry, University of Konstanz, D-78464 Konstanz, Germany. marina.rubini@uni-konstanz.de.
authors.forename: M
authors.initials: M
authors.lastname: Rubini
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D015394:Molecular Structure; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D004921:Erythropoietin; D011092:Polyethylene Glycols