Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli

International Journal of Pharmaceutics
Yin-Jue WangZhi-Guo Su

Abstract

Recombinant human erythropoietin produced by mammalian cells contains about 40% carbohydrates which maintain its stability and long residence in body. However, mammalian derived Epo has low yields and high costs of production. In this article, a cost-effective strategy of producing non-glycosylated Epo from Escherichia coli and then PEGylating it to replace the role of sugar chains was investigated. Recombinant human non-glycosylated erythropoietin (rh-ngEpo) was overexpressed as inclusion body in E. coli. As the routine inclusion body washing step resulted in poor protein recovery and purity, a new process scheme of using strong ion-exchange chromatography to purify denatured rh-ngEpo from inclusion body before refolding was developed. The purity of the denatured rh-ngEpo was increased from 59% to over 90%. Rh-ngEpo was then refolded and subsequently purified by one step of weak cation-exchange chromatography to 98% pure. Final protein yield was 129 mg/l, a significant improvement from 49 mg/l obtained via the conventional practice. The in vitro bioactivity of purified rh-ngEpo was comparable with the CHO-expressed Epo and the formation of native secondary structure was also confirmed by CD spectra. Rh-ngEpo was then modified ...Continue Reading

Citations

Jul 15, 2011·International Journal of Nanomedicine·Reza Ahangari CohanDariush Norouzian
Mar 12, 2013·Cellular and Molecular Life Sciences : CMLS·Samuel S NewtonRonald S Duman
Apr 30, 2010·Journal of Controlled Release : Official Journal of the Controlled Release Society·Yin-Jue WangZhi-Guo Su
Jan 15, 2014·Protein Expression and Purification·Taeck-Hyun JeongHan Choe
Nov 10, 2011·European Journal of Pharmaceutics and Biopharmaceutics : Official Journal of Arbeitsgemeinschaft Für Pharmazeutische Verfahrenstechnik E.V·Ahmad MalekiHosnieh Tajerzadeh
Nov 20, 2016·The Journal of Pharmacy and Pharmacology·Brittney J Mills, Jennifer S Laurence Chadwick
May 9, 2014·Bioconjugate Chemistry·Brittney J MillsJennifer S Laurence

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