Multistep purification of cytochrome c PEGylated forms using polymer-based aqueous biphasic systems

Santos, João H. P. M.; Carretero, Gustavo; Coutinho, João A. P.; Rangel-Yagui, Carlota de Oliveira; Ventura, Sónia P. M.

doi:10.1039/c7gc02600e

Cited by 18 publications

(13 citation statements)

References 61 publications

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“…The initial use of 2kDa mPEG was a proof of concept, since this reactive PEG is relatively cheaper. Additionally, a significant excess of mPEG-NHS was used due to its susceptibility to suffer hydrolysis in aqueous solutions; these values of molar ratio are in accordance with previous works [12,13,14]. Each system, at selected conditions, was kept under mechanical stirring at room temperature (22 to 25°C) for 30 minutes.…”

Section: Methodssupporting

confidence: 60%

Novel site-specific PEGylated L-asparaginase

et al. 2019

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L-asparaginase (ASNase) from Escherichia coli is currently used in some countries in its PEGylated form (ONCASPAR, pegaspargase) to treat acute lymphoblastic leukemia (ALL). PEGylation refers to the covalent attachment of poly(ethylene) glycol to the protein drug and it not only reduces the immune system activation but also decreases degradation by plasmatic proteases. However, pegaspargase is randomly PEGylated and, consequently, with a high degree of polydispersity in its final formulation. In this work we developed a site-specific N-terminus PEGylation protocol for ASNase. The monoPEG-ASNase was purified by anionic followed by size exclusion chromatography to a final purity of 99%. The highest yield of monoPEG-ASNase of 42% was obtained by the protein reaction with methoxy polyethylene glycol-carboxymethyl N -hydroxysuccinimidyl ester (10kDa) in 100 mM PBS at pH 7.5 and PEG:ASNase ratio of 25:1. The monoPEG-ASNase was found to maintain enzymatic stability for more days than ASNase, also was resistant to the plasma proteases like asparaginyl endopeptidase and cathepsin B. Additionally, monoPEG-ASNase was found to be potent against leukemic cell lines (MOLT-4 and REH) in vitro like polyPEG-ASNase. monoPEG-ASNase demonstrates its potential as a novel option for ALL treatment, being an inventive novelty that maintains the benefits of the current enzyme and solves challenges.

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Section: Methodssupporting

confidence: 60%

Novel site-specific PEGylated L-asparaginase

et al. 2019

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“…DTNB assay using 5,5′-dithio-bis-(2-nitrobenzoic acid) was performed to ensure the presence of free thiol prior to the conjugation reaction 33 . Cysteine-directed PEGylation was performed using thiol-maleimide chemistry 23 , 34 , to target the lone cysteine residue previously introduced by mutagenesis at position E18C. Purified fusion protein (2 mg/mL) was added to 20-fold molar excess of PEG-malemide (5 kDa) in presence of 50 mM Tris–Cl (pH 8).…”

Section: Methodsmentioning

confidence: 99%

“…AMP-polymer conjugates not only preserve their intrinsic antimicrobial activity, but also alleviate their toxicity and offers new functionalities 22 . Particularly, conjugation with poly(ethylene)glycol (PEG) has been extensively investigated for a range of therapeutic biomolecules to overcome the disadvantages associated with them 23 , 24 . There are limited reports on PEGylation of AMPs despite the fact that PEG conjugation offers important advantages such as improved solubility, reduced toxicity and decreased proteolytic susceptibility of these multifunctional molecules 25 , 26 .…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression, purification and PEGylation of Paneth cell peptide (cryptdin-2) with value added attributes against Staphylococcus aureus

Kaur

Dilawari

Kaur

et al. 2020

Sci Rep

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Cryptdins are disulfide-rich cationic antimicrobial peptides secreted by mouse Paneth cells and are known to exhibit potent antimicrobial activity against various deadly pathogens. Keeping in view the extremely low yield obtained from mouse Paneth cells and high cost of synthetic peptide(s), herein, we have attempted to produce cryptdin-2 in Escherichia coli using recombinant technology. To avoid lethal effects of peptide on the host cells, cryptdin-2 was expressed as a fusion protein with thioredoxin as fusion partner which yielded 40 mg/L protein in the soluble fraction. Subsequently, mature cryptdin-2 was cleaved from the fusion partner and purified by cation exchange chromatography. Since conjugation of poly(ethylene) glycol (PEG) has been known to improve the biological properties of biomolecules, therefore, we further attempted to prepare PEG-conjugated variant of cryptdin-2 using thiol specific PEGylation. Though the antimicrobial activity of PEGylated cryptdin-2 was compromised to some extent, but it was found to have enhanced serum stability for longer duration as compared to its un-modified forms. Also, it was found to exhibit reduced toxicity to the host cells. Further, its synergism with gentamicin suggests that PEGylated cryptdin-2 can be used with conventional antibiotics, thereby indicating its possibility to be used as an adjunct therapy.

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“…PEGylation reaction mixtures were purified by size exclusion chromatography (SEC) using a Superdex™ 200 Increase 10/300 GL column (crosslinked agarose–dextran resin) (Cytiva ® , Marlborough, MA, USA) in an AKTA™ purifier system (Cytiva ® , Marlborough, MA, USA) [ 22 ]. The column was equilibrated with 0.01 M phosphate buffer (0.14 M NaCl, pH 7.4) and eluted with the same buffer at a flow of 0.75 mL.min −1 .…”

Section: Methodsmentioning

confidence: 99%

Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability

et al. 2022

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Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30–40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C.

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Multistep purification of cytochrome c PEGylated forms using polymer-based aqueous biphasic systems

Abstract: An efficient process of PEGylation and purification of different site-specific Cyt-c PEGylated forms was developed with high efficiency using polymer-based aqueous biphasic systems.

Cited by 18 publications

References 61 publications

Novel site-specific PEGylated L-asparaginase

Novel site-specific PEGylated L-asparaginase

Recombinant expression, purification and PEGylation of Paneth cell peptide (cryptdin-2) with value added attributes against Staphylococcus aureus

Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability

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