Design, modeling, and expression of erythropoietin cysteine analogs in Pichia pastoris: Improvement of mean residence times and in vivo activities through cysteine-specific PEGylation

Ahmad, Mohd Ridzuan; Madadkar-Sobhani, Armin; Roohvand, Farzin; Najafabadi, Abdolhossein R.; Shafiee, Abbas; Khanahmad, Hossein; Cohan, Reza Ahangari; Namvar, N A; Tajerzadeh, Hosnieh

doi:10.1016/j.ejpb.2011.10.017

Cited by 12 publications

(6 citation statements)

References 42 publications

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“…It should be noted that the possible sites for the conjugation (even on consideration of experimental information in the literature) are very numerous. 11 , 15 The condition is more complicated with variation in PEG sizes and shapes that could employed for PEGylation. 16 , 17 Therefore, the examination of all possible sites is not practically achievable in a lab as it requires considerable time and cost.…”

Section: Discussionmentioning

confidence: 99%

Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins

Mirzaei

Kazemi

Bandehpour

et al. 2016

DDDT

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Cysteine PEGylation includes several steps, and is difficult to manage in practice. In the current investigation, the cysteine PEGylation of erythropoietin analogs was examined using computational and nonglycosylated systems to define a simpler approach for specific PEGylation. Two model analogs (E31C and E89C) were selected for PEGylation based on lowest structural deviation from the native form, accessibility, and nucleophilicity of the free thiol group. The selected analogs were cloned and the expression was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot using Coomassie blue staining and anti-His monoclonal antibody, respectively. PEGylation with 20 kDa mPEG-maleimide resulted in 79% and 82% conjugation yield for E31C and E89C nonglycosylated erythropoietin (ngEPO) analogs, respectively. The size distribution and charge analysis showed an increase in size and negative charge of the PEGylated forms compared with nonconjugated ones. Biological assay revealed that E31C and E89C mutations and subsequent PEGylation of ngEPO analogs have no deleterious effects on in vitro biological activity when compared to CHO-derived recombinant human erythropoietin. In addition, PEG-conjugated ngEPOs showed a significant increase in plasma half-lives after injection into rats when compared to nonconjugated ones. The development of the cysteine-PEGylated proteins using nonglycosylated expression system and in silico technique can be considered an efficient approach in terms of optimization of PEGylation parameters, time, and cost.

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Section: Discussionmentioning

confidence: 99%

Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins

Mirzaei

Kazemi

Bandehpour

et al. 2016

DDDT

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“…The mean area under the curve (AUC 02' ) was determined using a linear trapezoidal method with extrapolation to infinity. Clearance (CL) and volume of distribution (V d ) values were also calculated (Cohan et al, 2011;Maleki et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

New Proline, Alanine, Serine Repeat Sequence for Pharmacokinetic Enhancement of Anti-VEGF Single-Domain Antibody

Khodabakhsh

Salimian

Mehdizadeh

et al. 2020

J Pharmacol Exp Ther

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Therapeutic fragmented antibodies show a poor pharmacokinetic profile that leads to frequent high-dose administration. In the current study, for the first time, a novel proline, alanine, serine (PAS) repeat sequence called PAS#208 was designed to extend the plasma half-life of a nanosized anti-vascular endothelial growth factor-A single-domain antibody. Polyacrylamide gel electrophoresis, circular dichroism, dynamic light scattering, and electrophoretic light scattering were used to assess the physicochemical properties of the newly designed PAS sequence. The effect of PAS#208 on the biologic activity of a single-domain antibody was studied using an in vitro proliferation assay. The pharmacokinetic parameters, including terminal half-life, the volume of distribution, elimination rate constant, and clearance, were determined in mice model and compared with the native protein and PAS#1(200) sequence. The novel PAS repeat sequence showed comparable physicochemical, biologic, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The PAS#208 increased the hydrodynamic radius and decreased significantly the electrophoretic mobility of the native protein without any change in zeta potential. Surprisingly, the fusion of PAS#208 to the single-domain antibody increased the binding potency. In addition, it did not alter the biologic activity and did not show any cytotoxicity on the normal cells. The PAS#208 sequence improved the terminal half-life (14-fold) as well as other pharmacokinetic parameters significantly. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of therapeutic fragmented antibodies. SIGNIFICANCE STATEMENTIn the current study, a new proline, alanine, serine (PAS) sequence was developed that showed comparable physicochemical, biological, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of recombinant small proteins.

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“…Cysteine residue is amongst the most used groups for protein conjugation strategies. As a result of the absence of a free thiol in the native proteins, engineered cysteine residues can be introduced by genetic mutagenesis . The approach allows well defined site‐specific modification on the proteins that can minimize the potential loss in biological activity.…”

Section: Introductionmentioning

confidence: 99%

Protein purification by chemo‐selective precipitation using thermoresponsive polymers

Cai

et al. 2018

Biopolymers

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A recoverable and thermoresponsive polymer-protein bioconjugate is synthesized and employed in the purification of protein with free sulfhydryl groups. Initiator with disulphide was modified on the cysteine residue of the target protein. Poly(N-isopropylacrylamide) exhibiting a lower critical solution temperature was grown from the protein. The resulting protein-polymer conjugate was successfully thermoprecipitated and separated from other proteins. The approach was demonstrated with bovine serum albumin with the recycling yield of 76.4%. Enzyme activity test with papain verified the reversible polymer modification protected protein under extreme environments without affecting the functionality of the protein. This study implies the favorable potential of chemo-selective enriching and purification of proteins.

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Design, modeling, and expression of erythropoietin cysteine analogs in Pichia pastoris: Improvement of mean residence times and in vivo activities through cysteine-specific PEGylation

Cited by 12 publications

References 42 publications

Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins

Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins

New Proline, Alanine, Serine Repeat Sequence for Pharmacokinetic Enhancement of Anti-VEGF Single-Domain Antibody

Protein purification by chemo‐selective precipitation using thermoresponsive polymers

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