Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites

E. Hoffmann; K. Streichert; N. Nischan; C. Seitz; T. Brunner; S. Schwagerus; C. P. R. Hackenberger; M. Rubini

doi:10.1039/C5MB00857C

Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites†

E. Hoffmann,^a K. Streichert,^b N. Nischan,‡^b C. Seitz,^c T. Brunner,^c S. Schwagerus,^b C. P. R. Hackenberger*^b and M. Rubini*^a

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* Corresponding authors

^a Department of Organic Chemistry, University of Konstanz, D-78464 Konstanz, Germany
E-mail: marina.rubini@uni-konstanz.de
Fax: +49 7531884150
Tel: +49 7531 882398

^b Leibniz Institute of Molecular Pharmacology, D-13125 Berlin and Humboldt Universität zu Berlin, D-12489 Berlin, Germany
E-mail: hackenbe@fmp-berlin.de
Fax: +49 30 94793109
Tel: +49 30 94793181

^c Department of Biochemical Pharmacology, University of Konstanz, D-78464 Konstanz, Germany

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.

This article is part of the themed collections: Chemical Biology in Molecular BioSystems and Protein Labelling

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DOI: https://doi.org/10.1039/C5MB00857C
Article type: Communication
Submitted: 09 Dec 2015
Accepted: 09 Jan 2016
First published: 11 Jan 2016
This article is Open Access

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Mol. BioSyst., 2016,12, 1750-1755

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Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites

E. Hoffmann, K. Streichert, N. Nischan, C. Seitz, T. Brunner, S. Schwagerus, C. P. R. Hackenberger and M. Rubini, Mol. BioSyst., 2016, 12, 1750 DOI: 10.1039/C5MB00857C

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Molecular BioSystems

Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites†

Abstract

Supplementary files

Article information

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Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites

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