Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

Reza Ahangari Cohan1, Armin Madadkar-Sobhani2,3, Hossein Khanahmad1, Farzin Roohvand4, Mohammad Reza Aghasadeghi4, Mohammad Hossein Hedayati5, Zahra Barghi5, Mehdi Shafiee Ardestani4, Davoud Nouri Inanlou1, Dariush Norouzian11Research and Development Department, Production and Research Complex, Past...

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Autores principales:	Ahangari Cohan R, Madadkar-Sobhani A, Khanahmad H, Roohv, F, Aghasadeghi MR, Hedayati MH, Barghi Z, Shafiee Ardestani M, Nouri Inanlou D, Norouzian D
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2011
Materias:	Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/8990c8a908714a4c93006f694d5d193c
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spelling	oai:doaj.org-article:8990c8a908714a4c93006f694d5d193c2021-12-02T05:02:11ZDesign, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin1176-91141178-2013https://doaj.org/article/8990c8a908714a4c93006f694d5d193c2011-06-01T00:00:00Zhttp://www.dovepress.com/design-modeling-expression-and-chemoselective-pegylation-of-a-new-nano-a7657https://doaj.org/toc/1176-9114https://doaj.org/toc/1178-2013Reza Ahangari Cohan1, Armin Madadkar-Sobhani2,3, Hossein Khanahmad1, Farzin Roohvand4, Mohammad Reza Aghasadeghi4, Mohammad Hossein Hedayati5, Zahra Barghi5, Mehdi Shafiee Ardestani4, Davoud Nouri Inanlou1, Dariush Norouzian11Research and Development Department, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran; 2Department of Bioinformatics, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; 3Department of Life Sciences, Barcelona Supercomputing Center, Barcelona, Spain; 4Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran; 5Quality Control Department, Production and Research Complex, Pasteur Institute of Iran, Tehran, IranBackground: 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.Discussion: This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.Keywords: nanoPEGylated EPO, cysteine PEGylation, pharmacokinetic propertyAhangari Cohan RMadadkar-Sobhani AKhanahmad HRoohvFAghasadeghi MRHedayati MHBarghi ZShafiee Ardestani MNouri Inanlou DNorouzian DDove Medical PressarticleMedicine (General)R5-920ENInternational Journal of Nanomedicine, Vol 2011, Iss default, Pp 1217-1227 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine (General) R5-920
spellingShingle	Medicine (General) R5-920 Ahangari Cohan R Madadkar-Sobhani A Khanahmad H Roohv F Aghasadeghi MR Hedayati MH Barghi Z Shafiee Ardestani M Nouri Inanlou D Norouzian D Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin
description	Reza Ahangari Cohan1, Armin Madadkar-Sobhani2,3, Hossein Khanahmad1, Farzin Roohvand4, Mohammad Reza Aghasadeghi4, Mohammad Hossein Hedayati5, Zahra Barghi5, Mehdi Shafiee Ardestani4, Davoud Nouri Inanlou1, Dariush Norouzian11Research and Development Department, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran; 2Department of Bioinformatics, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; 3Department of Life Sciences, Barcelona Supercomputing Center, Barcelona, Spain; 4Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran; 5Quality Control Department, Production and Research Complex, Pasteur Institute of Iran, Tehran, IranBackground: 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.Discussion: This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.Keywords: nanoPEGylated EPO, cysteine PEGylation, pharmacokinetic property
format	article
author	Ahangari Cohan R Madadkar-Sobhani A Khanahmad H Roohv F Aghasadeghi MR Hedayati MH Barghi Z Shafiee Ardestani M Nouri Inanlou D Norouzian D
author_facet	Ahangari Cohan R Madadkar-Sobhani A Khanahmad H Roohv F Aghasadeghi MR Hedayati MH Barghi Z Shafiee Ardestani M Nouri Inanlou D Norouzian D
author_sort	Ahangari Cohan R
title	Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin
title_short	Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin
title_full	Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin
title_fullStr	Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin
title_full_unstemmed	Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin
title_sort	design, modeling, expression, and chemoselective pegylation of a new nanosize cysteine analog of erythropoietin
publisher	Dove Medical Press
publishDate	2011
url	https://doaj.org/article/8990c8a908714a4c93006f694d5d193c
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Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

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