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Use of Cross-Linked Poly(ethylene glycol)-Based Hydrogels for Protein Crystallization.

Gavira JA, Cera-Manjarres A, Ortiz K, Mendez J, Jimenez-Torres JA, Patiño-Lopez LD, Torres-Lugo M - Cryst Growth Des (2014)

Bottom Line: PEG hydrogels also induced the nucleation of lysozyme crystals to a higher extent than agarose.As an example, insulin crystals were grown in 10% (w/w) PEG hydrogel.The resulting crystals were of an approximate size of 500 μm.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Estudios Crystalográficos, IACT (CSIC-UGR). Avda. las Palmeras 4, E18100 Armilla, Granada, Spain.

ABSTRACT
Poly(ethylene glycol) (PEG) hydrogels are highly biocompatible materials extensively used for biomedical and pharmaceutical applications, controlled drug release, and tissue engineering. In this work, PEG cross-linked hydrogels, synthesized under various conditions, were used to grow lysozyme crystals by the counterdiffusion technique. Crystallization experiments were conducted using a three-layer arrangement. Results demonstrated that PEG fibers were incorporated within lysozyme crystals controlling the final crystal shape. PEG hydrogels also induced the nucleation of lysozyme crystals to a higher extent than agarose. PEG hydrogels can also be used at higher concentrations (20-50% w/w) as a separation chamber (plug) in counterdiffusion experiments. In this case, PEG hydrogels control the diffusion of the crystallization agent and therefore may be used to tailor the supersaturation to fine-tune crystal size. As an example, insulin crystals were grown in 10% (w/w) PEG hydrogel. The resulting crystals were of an approximate size of 500 μm.

No MeSH data available.


Related in: MedlinePlus

Slope of the linear curve fit to the nucleation front positionvs the square root of time, for each PEG plug composition (gel poresize is indicated in the right axes). This slope is a measure of thenucleation front rate in 3L counterdiffusion experiments. Errorbars for the slope represent a 95% confidence interval. Error barsfor the mesh size represent one standard deviation from the averagevalue.
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fig4: Slope of the linear curve fit to the nucleation front positionvs the square root of time, for each PEG plug composition (gel poresize is indicated in the right axes). This slope is a measure of thenucleation front rate in 3L counterdiffusion experiments. Errorbars for the slope represent a 95% confidence interval. Error barsfor the mesh size represent one standard deviation from the averagevalue.

Mentions: The nucleation process began approximately12 h after the addition of the crystallizing agent. All experimentsdemonstrated a typical counterdiffusion pattern, with high nucleationdensity near the plug, which decreases along the protein chamber,resulting in a lower number of crystals of larger size at the endof the tube. The advancement of the nucleation front was measuredwith respect to time. The nucleation front position was plotted againstthe square root of time, and in all cases, it produced linear trends,which indicated that the transport of the crystallization agent wasdominated by passive diffusion following Fick’s second law.31 Therefore, the slope of the linear curve fittingis a measure of the rate at which the nucleation front is moving alongthe protein chamber. The results depicted in Figure 4 illustrate the nucleation front rate along the protein chamber,that is, the slope of the linear curve fit, as a function of gel concentration(pore size). As expected, the nucleation rate decreased as the gel–plugconcentration increased. Although in all cases crystals of millimetersize were obtained at the end of the protein chamber, results indicatedthat the 50% (w/w) dilution PEG hydrogel plugs rendered crystals slightlylarger when compared to experiments with 20 or 35% (w/w) plugs (FigureS3).


Use of Cross-Linked Poly(ethylene glycol)-Based Hydrogels for Protein Crystallization.

Gavira JA, Cera-Manjarres A, Ortiz K, Mendez J, Jimenez-Torres JA, Patiño-Lopez LD, Torres-Lugo M - Cryst Growth Des (2014)

Slope of the linear curve fit to the nucleation front positionvs the square root of time, for each PEG plug composition (gel poresize is indicated in the right axes). This slope is a measure of thenucleation front rate in 3L counterdiffusion experiments. Errorbars for the slope represent a 95% confidence interval. Error barsfor the mesh size represent one standard deviation from the averagevalue.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215911&req=5

fig4: Slope of the linear curve fit to the nucleation front positionvs the square root of time, for each PEG plug composition (gel poresize is indicated in the right axes). This slope is a measure of thenucleation front rate in 3L counterdiffusion experiments. Errorbars for the slope represent a 95% confidence interval. Error barsfor the mesh size represent one standard deviation from the averagevalue.
Mentions: The nucleation process began approximately12 h after the addition of the crystallizing agent. All experimentsdemonstrated a typical counterdiffusion pattern, with high nucleationdensity near the plug, which decreases along the protein chamber,resulting in a lower number of crystals of larger size at the endof the tube. The advancement of the nucleation front was measuredwith respect to time. The nucleation front position was plotted againstthe square root of time, and in all cases, it produced linear trends,which indicated that the transport of the crystallization agent wasdominated by passive diffusion following Fick’s second law.31 Therefore, the slope of the linear curve fittingis a measure of the rate at which the nucleation front is moving alongthe protein chamber. The results depicted in Figure 4 illustrate the nucleation front rate along the protein chamber,that is, the slope of the linear curve fit, as a function of gel concentration(pore size). As expected, the nucleation rate decreased as the gel–plugconcentration increased. Although in all cases crystals of millimetersize were obtained at the end of the protein chamber, results indicatedthat the 50% (w/w) dilution PEG hydrogel plugs rendered crystals slightlylarger when compared to experiments with 20 or 35% (w/w) plugs (FigureS3).

Bottom Line: PEG hydrogels also induced the nucleation of lysozyme crystals to a higher extent than agarose.As an example, insulin crystals were grown in 10% (w/w) PEG hydrogel.The resulting crystals were of an approximate size of 500 μm.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Estudios Crystalográficos, IACT (CSIC-UGR). Avda. las Palmeras 4, E18100 Armilla, Granada, Spain.

ABSTRACT
Poly(ethylene glycol) (PEG) hydrogels are highly biocompatible materials extensively used for biomedical and pharmaceutical applications, controlled drug release, and tissue engineering. In this work, PEG cross-linked hydrogels, synthesized under various conditions, were used to grow lysozyme crystals by the counterdiffusion technique. Crystallization experiments were conducted using a three-layer arrangement. Results demonstrated that PEG fibers were incorporated within lysozyme crystals controlling the final crystal shape. PEG hydrogels also induced the nucleation of lysozyme crystals to a higher extent than agarose. PEG hydrogels can also be used at higher concentrations (20-50% w/w) as a separation chamber (plug) in counterdiffusion experiments. In this case, PEG hydrogels control the diffusion of the crystallization agent and therefore may be used to tailor the supersaturation to fine-tune crystal size. As an example, insulin crystals were grown in 10% (w/w) PEG hydrogel. The resulting crystals were of an approximate size of 500 μm.

No MeSH data available.


Related in: MedlinePlus