title: Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin.
abstract: BACKGROUND
  Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem.
  
  
  METHODS
  A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr(-) cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized.
  
  
  RESULTS
  The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement.
  
  
  CONCLUSIONS
  This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.
authors.affiliation: Research and Development Department, Production and Research Complex, Pasteur Institute of Iran, Tehran.
authors.forename: Reza Ahangari
authors.initials: RA
authors.lastname: Cohan
authors.forename: Armin
authors.initials: A
authors.lastname: Madadkar-Sobhani
authors.forename: Hossein
authors.initials: H
authors.lastname: Khanahmad
authors.forename: Farzin
authors.initials: F
authors.lastname: Roohvand
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Aghasadeghi
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Hedayati
authors.forename: Zahra
authors.initials: Z
authors.lastname: Barghi
authors.forename: Mehdi Shafiee
authors.initials: MS
authors.lastname: Ardestani
authors.forename: Davoud Nouri
authors.initials: DN
authors.lastname: Inanlou
authors.forename: Dariush
authors.initials: D
authors.lastname: Norouzian
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D049109:Cell Proliferation; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016503:Drug Delivery Systems; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D053758:Nanoparticles / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: C554270:EPO protein, human; D011994:Recombinant Proteins; D004921:Erythropoietin; D018698:Glutamic Acid; D011092:Polyethylene Glycols; D003545:Cysteine; D008727:Methotrexate