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

Abstract

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.

Effects and treatment applications of polymeric nanoparticles on improving platelets' storage time: a review of the literature from 2010 to 2020

“…Mirzaei et al . [ 67 ] linked ALGD to the antibody and showed that the nanoparticles were capable of binding to the protein and reducing the toxicity of the antibody. They PEGylated erythropoietin analogs using ALGDs and investigated their properties in vitro .…”

From Synthesis to Characterization of Site-Selective PEGylated Proteins

“…The results suggested that the PEG polymer wraps around the protein providing steric shield, and this effect depends on the PEG chain length and PEGylation site of the protein (Rouhani et al, 2018b). Also, Mirzaei et al (2016) applied computational and non-glycosylated systems to define an artless methodology for site-selective (cysteine) PEGylation of erythropoietin analogs. The results showed that using an in silico approach together with the experimental methodologies can be a strategy to optimize the parameters of PEGylation (Mirzaei et al, 2016).…”

“…Also, Mirzaei et al (2016) applied computational and non-glycosylated systems to define an artless methodology for site-selective (cysteine) PEGylation of erythropoietin analogs. The results showed that using an in silico approach together with the experimental methodologies can be a strategy to optimize the parameters of PEGylation (Mirzaei et al, 2016). Recently, Xu et al (2018) used interferon (IFN) as a representative model system to characterize the molecular-level changes in IFN introduced by several degrees of PEGylation through molecular dynamics simulations.…”

Potential of MMP-2 and MMP-9 Gelatinase Blockade as a Therapeutic Strategy in Fibrosarcoma Treatment: A Decadal Review

“…These molecules can be engineered to carry out a variety of functions, such as directly inhibiting tumor growth, blocking signals that promote cancer cell survival, or delivering cytotoxic agents specifically to cancer cells [18,105]. Compared to traditional chemotherapy, peptide and protein therapies often have lower toxicity, leading to fewer and less severe side effects [106]. Being natural biological molecules, proteins and peptides are biodegradable and less likely to accumulate in the body, reducing long-term side effects [107].…”