title: Design, modeling, and expression of erythropoietin cysteine analogs in Pichia pastoris: improvement of mean residence times and in vivo activities through cysteine-specific PEGylation.
abstract: In this study, the low-cost production of recombinant human erythropoietin cysteine analogs (Cys-rhEPOs) from Pichia pastoris and the potential to increase their serum residency and in vivo activity through cysteine-specific PEGylation were investigated. Three-dimensional structures of several Cys-rhEPOs were generated using homology modeling, and three stable Cys-rhEPOs were selected on the basis of model stability in molecular dynamics simulation and surface accessibility of the inserted cysteine. cDNAs encoding Cys-rhEPOs were constructed by site-directed mutagenesis and expressed as secreted proteins in flask cultures of P. pastoris. The selection of highly expressing clones and the optimization of certain culture parameters resulted in protein expression levels of 100-170 mg/l. Purified Cys-rhEPOs were cysteine-specifically PEGylated using 20 kDa and 30 kDa mPEG-maleimides (methoxy polyethylene glycol-maleimides). The E89CEPO analog with the highest (96.6%) cysteine accessibility was conjugated to PEG-polymers with the largest yields (about 80%). In comparison with rhEPO, 30 kDa PEG-E89CEPO demonstrated a significant (approximately 30%) increase in the mean residence time. Whereas the in vitro activities of 30 kDa PEG-E89CEPO were comparable to those of rhEPO, the in vivo activity of this conjugate was more prolonged compared to rhEPO (12 days vs. 7 days). Our results demonstrate that the site-specific PEGylation of Pichia-expressed EPO analogs may be considered as a promising approach for generating cost-effective and long-acting erythropoiesis-stimulating agents.
authors: Department of Pharmaceutics, The Tehran University of Medical Science, Tehran, Iran.
authors: Ahmad
authors: A
authors: Maleki
authors: Armin
authors: A
authors: Madadkar-Sobhani
authors: Farzin
authors: F
authors: Roohvand
authors: Abdolhossein R
authors: AR
authors: Najafabadi
authors: Abbas
authors: A
authors: Shafiee
authors: Hossein
authors: H
authors: Khanahmad
authors: Reza A
authors: RA
authors: Cohan
authors: Nabiallah
authors: N
authors: Namvar
authors: Hosnieh
authors: H
authors: Tajerzadeh
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D019295:Computational Biology / Q000379:methods; D003545:Cysteine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics; D015195:Drug Design; D004921:Erythropoietin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006397:Hematinics / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008297:Male; D008301:Maleimides / Q000737:chemistry; D051379:Mice; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D006397:Hematinics; D008301:Maleimides; D011994:Recombinant Proteins; D004921:Erythropoietin; D011092:Polyethylene Glycols; D003545:Cysteine