title: Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli.
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 by a 20 kDa methoxy polyethylene glycol (PEG) succinimidyl carbonate. The monoPEGylated protein, which retained 68% bioactivity, had enhanced thermal stability and a remarkably prolonged circulating half-life in rats as compared with that of the unmodified protein. These studies demonstrated the feasibility of PEGylating rh-ngEpo as a promising way for the development of new Epo drugs.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China.
authors.forename: Yin-Jue
authors.initials: YJ
authors.lastname: Wang
authors.forename: Yong-Dong
authors.initials: YD
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Su-Juan
authors.initials: SJ
authors.lastname: Hao
authors.forename: Tao
authors.initials: T
authors.lastname: Hu
authors.forename: Guang-Hui
authors.initials: GH
authors.lastname: Ma
authors.forename: Zhi-Guo
authors.initials: ZG
authors.lastname: Su
mesh_terms: D000818:Animals; D016466:CHO Cells; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000008:administration & dosage / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000493:pharmacokinetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005240:Feasibility Studies; D006031:Glycosylation; D006207:Half-Life; D006397:Hematinics / Q000008:administration & dosage / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000493:pharmacokinetics; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D007279:Injections, Subcutaneous; D008297:Male; D011092:Polyethylene Glycols / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D013388:Succinimides / Q000737:chemistry*; D013678:Technology, Pharmaceutical / Q000379:methods; D013696:Temperature
chemical_list: D002411:Cation Exchange Resins; D006397:Hematinics; D011994:Recombinant Proteins; D013388:Succinimides; D004921:Erythropoietin; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol