Evaluation of PEG/phosphate aqueous two-phase systems for the purification of the chicken egg white protein avidin by using high-throughput techniques

Diederich, Patrick; Amrhein, Sven; Hämmerling, Frank; Hubbuch, Jürgen

doi:10.1016/j.ces.2013.10.008

Cited by 26 publications

(25 citation statements)

References 32 publications

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“…Two dissimilar polymers that interact unfavorably phase separate even at low concentrations of polymer solute. 49, 50, 100, 101 Aqueous mixtures of PEG and protein have been widely studied owing to their relevance to protein crystallization, 51, 52 separation/purification strategies, 53–55 and the analysis of disease progression. 97 However, most protein-PEG solutions eventually form macroscopic phases; 49, 97 in the RLP-PEG hydrogels, the cross-linking reaction competes with the process of macroscale phase separation and effectively captures heterogeneous micron-size domains of different compositions.…”

Section: Resultsmentioning

confidence: 99%

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure

McGann¹,

Akins

Kiick

2015

Biomacromolecules

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Hydrogels derived from resilin-like polypeptides (RLPs) have shown outstanding mechanical resilience and cytocompatibility, and expanding the versatility of RLP-based materials via conjugation with other polypeptides and polymers would offer great promise in the design of a range of materials. Here, we present an investigation of the biochemical and mechanical properties of hybrid hydrogels composed of a recombinant RLP and a multi-arm PEG macromer. These hybrid hydrogels can be rapidly cross-linked through a Michael-type addition reaction between the thiols of cysteine residues on the RLP and vinyl sulfone groups on the multi-arm PEG. Oscillatory rheology and tensile testing confirmed the formation of elastomeric hydrogels with mechanical resilience comparable to aortic elastin; hydrogel stiffness was easily modulated through the cross-linking ratio. Macromolecular phase separation of the RLP-PEG hydrogels offers the unique advantage of imparting a heterogeneous microstructure, which can be used to localize cells, through simple mixing and crosslinking. Assessment of degradation of the RLP by matrix metalloproteinases (MMPs) illustrated the specific proteolysis of the polypeptide in both its soluble form and when cross-linked into hydrogels. Finally, the successful encapsulation and viable three-dimensional culture of human mesenchymal stem cells (hMSCs) demonstrated the cytocompatibility of the RLP-PEG gels. Overall, the cytocompatibility, elastomeric mechanical properties, micro-heterogeneity, and degradability of the RLP-PEG hybrid hydrogels offer a suite of promising properties for the development of cell-instructive, structured tissue engineering scaffolds.

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Section: Resultsmentioning

confidence: 99%

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure

McGann¹,

Akins

Kiick

2015

Biomacromolecules

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“…Yang and co-workers [25] demonstrated the application of PEG 4000/potassium citrate ABS to extract lysozyme from egg white. Diederich et al [26] showed the selective separation of avidin from the remaining proteins from egg white using an ABS composed of PEG 600 + potassium phosphate + 3 wt% NaCl, with a purity level higher than 60% and recovery yields > 90% (for avidin). In an attempt to reach higher purification levels, multi-stage ABS separations, by means of liquid-liquid chromatography, have been also investigated.…”

Section: Introductionmentioning

confidence: 99%

Single-step purification of ovalbumin from egg white using aqueous biphasic systems

Pereira

Cruz

Almeida

et al. 2016

Process Biochemistry

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The ability of aqueous biphasic systems (ABS) composed of polyethylene glycols of different molecular weights (PEG 400, 600 and 1000) and buffered aqueous solutions of potassium citrate/ citric acid (pH = 5.0 -8.0) to selectively extract ovalbumin from egg white was here investigated. Phase diagrams, tie-lines and tie-line lengths were determined at 25ºC and the partitioning of ovalbumin in these systems was then evaluated. Aiming at optimizing the selective extraction of ovalbumin in the studied ABS, factors such as pH, PEG molecular weight and amount of the phase-forming components were initially investigated with pure commercial ovalbumin. In almost all ABS, it was observed a preferential partitioning of ovalbumin to the polymer-rich phase, with extraction efficiencies higher than 90%. The best ABS were then applied in the purification of ovalbumin from the real egg white matrix. In order to ascertain on the ovalbumin purity and yield, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high performance liquid chromatography (SE-HPLC) analyses were conducted, confirming that the isolation/purification of ovalbumin from egg white was completely achieved in a single-step with a recovery yield of 65%. The results obtained show that polymer-salt-based ABS allow the selective extraction of ovalbumin from egg white with a simpler approach and better performance than previously reported. Finally, it is shown that ovalbumin can be completely recovered from the PEG-rich phase by an induced precipitation using an inexpensive and sustainable separation platform which can be easily applied on an industrial scale.

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“…Furthermore, above 2 wt.% NaCl the partition behavior of BSA/OVA is highly dependent on increasing hydrophobic protein-PEG interactions and totally independent of the surface net charge, thus increasing K [1,70,82]. Additionally, the protein shift from the bottom into the top phase occurs due to an increasing salting-out effect in the salt-rich bottom phase as well as a rising shielding of the proteins' surface charges caused by Na + and Cl − ions [13,84]. Furthermore, NaCl has the ability of modifying the ordered water structure around the hydrophobic chain, like the ethylene group of PEG or the hydrophobic protein surface area exposed to solvent [70,85,86].…”

Section: Effect Of Nacl Concentrationmentioning

confidence: 99%

Evaluation of Driving Forces for Protein Partition in PEG-Salt Aqueous Two- Phase Systems and Optimization by Design of Experiments

Glyk¹,

Solle²,

Scheper³

et al. 2016

J Chromatogr Sep Tech

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Evaluation of PEG/phosphate aqueous two-phase systems for the purification of the chicken egg white protein avidin by using high-throughput techniques

Cited by 26 publications

References 32 publications

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure

Single-step purification of ovalbumin from egg white using aqueous biphasic systems

Evaluation of Driving Forces for Protein Partition in PEG-Salt Aqueous Two- Phase Systems and Optimization by Design of Experiments

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