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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

(A) Observable nucleation front position as a functionof the squareroot of time of 3L counterdiffusion experiments. The crystallizationchamber contains protein, in 10% (w/w) PEG cross-linked hydrogel or0.2% (w/v) agarose, at three concentrations. Error bars for the observablenucleation front position vs the square root of time represent onestandard deviation from the average of three independent experiments.Lines (dotted for agarose experiments) represent the linear curvefit. The slope of the fit, which gives an idea of the nucleation frontrate, is plotted in B vs protein concentration for the six types ofexperiments. Error bars in the slope vs protein concentration representthe 95% confidence interval.
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fig6: (A) Observable nucleation front position as a functionof the squareroot of time of 3L counterdiffusion experiments. The crystallizationchamber contains protein, in 10% (w/w) PEG cross-linked hydrogel or0.2% (w/v) agarose, at three concentrations. Error bars for the observablenucleation front position vs the square root of time represent onestandard deviation from the average of three independent experiments.Lines (dotted for agarose experiments) represent the linear curvefit. The slope of the fit, which gives an idea of the nucleation frontrate, is plotted in B vs protein concentration for the six types ofexperiments. Error bars in the slope vs protein concentration representthe 95% confidence interval.

Mentions: Figure 6A demonstrates the evolution ofthe observable nucleation front as a function of the square root oftime for each protein concentration. This representation allows usto conclude that a diffusional mass transport process controls thenucleation front evolution within the selected time frame since itfollows the analytical solution of Fick’s law.31 Therefore, the slope of the linear curve fitting is a measureof the rate at which the nucleation front is moving along the proteinchamber (Figure 6B). Experiments conductedin agarose evolved as expected; the nucleation front moved fasteras the protein concentration was increased. Also, the three experimentalseries (40, 50, and 60 mg/mL) performed with agarose started at almostthe same time with a small delay of the experiments performed with40 mg/mL lysozyme. These results indicated that the plug pore size(50% (w/w) PEG) controls the diffusion of the crystallization solution,and, therefore, determines the starting time. This behavior was notfollowed for the experiments conducted with PEG hydrogel. A closerlook at the evolution of the experiments (Figure 6A) indicated that the experiments performed using the PEGhydrogel started to nucleate earlier than the experiments conductedin agarose. Taking into account that agarose acts as a nucleationinducer,39 the PEG hydrogel is acting asa crystallization agent, enhancing the local supersaturationreached by the diffusion of sodium chloride. Although this effectis observed for all PEG hydrogel experiments, at 60 mg/mL of lysozymea delay can be observed. We believed that at high protein concentration,over 50 mg/mL, the interaction between the protein molecules and thePEG monomers might affect the polymerization, and protein could beentrapped within the gel network or even produce some protein denaturation.Any of these effects could lower the supersaturation when comparedwith the other two experimental series. Second, in all experimentsperformed with PEG-based hydrogel, the nucleation front advanced ata constant rate (slope) independently of the protein concentration(Figure 6B and Table S1). This observed behaviorpoints to an effect of the PEG pore diameter, much smaller than agarosepore size. We hypothesize that the PEG hydrogel is playing an essentialrole on the control of supersaturation by limiting the diffusionof protein molecules.


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)

(A) Observable nucleation front position as a functionof the squareroot of time of 3L counterdiffusion experiments. The crystallizationchamber contains protein, in 10% (w/w) PEG cross-linked hydrogel or0.2% (w/v) agarose, at three concentrations. Error bars for the observablenucleation front position vs the square root of time represent onestandard deviation from the average of three independent experiments.Lines (dotted for agarose experiments) represent the linear curvefit. The slope of the fit, which gives an idea of the nucleation frontrate, is plotted in B vs protein concentration for the six types ofexperiments. Error bars in the slope vs protein concentration representthe 95% confidence interval.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: (A) Observable nucleation front position as a functionof the squareroot of time of 3L counterdiffusion experiments. The crystallizationchamber contains protein, in 10% (w/w) PEG cross-linked hydrogel or0.2% (w/v) agarose, at three concentrations. Error bars for the observablenucleation front position vs the square root of time represent onestandard deviation from the average of three independent experiments.Lines (dotted for agarose experiments) represent the linear curvefit. The slope of the fit, which gives an idea of the nucleation frontrate, is plotted in B vs protein concentration for the six types ofexperiments. Error bars in the slope vs protein concentration representthe 95% confidence interval.
Mentions: Figure 6A demonstrates the evolution ofthe observable nucleation front as a function of the square root oftime for each protein concentration. This representation allows usto conclude that a diffusional mass transport process controls thenucleation front evolution within the selected time frame since itfollows the analytical solution of Fick’s law.31 Therefore, the slope of the linear curve fitting is a measureof the rate at which the nucleation front is moving along the proteinchamber (Figure 6B). Experiments conductedin agarose evolved as expected; the nucleation front moved fasteras the protein concentration was increased. Also, the three experimentalseries (40, 50, and 60 mg/mL) performed with agarose started at almostthe same time with a small delay of the experiments performed with40 mg/mL lysozyme. These results indicated that the plug pore size(50% (w/w) PEG) controls the diffusion of the crystallization solution,and, therefore, determines the starting time. This behavior was notfollowed for the experiments conducted with PEG hydrogel. A closerlook at the evolution of the experiments (Figure 6A) indicated that the experiments performed using the PEGhydrogel started to nucleate earlier than the experiments conductedin agarose. Taking into account that agarose acts as a nucleationinducer,39 the PEG hydrogel is acting asa crystallization agent, enhancing the local supersaturationreached by the diffusion of sodium chloride. Although this effectis observed for all PEG hydrogel experiments, at 60 mg/mL of lysozymea delay can be observed. We believed that at high protein concentration,over 50 mg/mL, the interaction between the protein molecules and thePEG monomers might affect the polymerization, and protein could beentrapped within the gel network or even produce some protein denaturation.Any of these effects could lower the supersaturation when comparedwith the other two experimental series. Second, in all experimentsperformed with PEG-based hydrogel, the nucleation front advanced ata constant rate (slope) independently of the protein concentration(Figure 6B and Table S1). This observed behaviorpoints to an effect of the PEG pore diameter, much smaller than agarosepore size. We hypothesize that the PEG hydrogel is playing an essentialrole on the control of supersaturation by limiting the diffusionof protein molecules.

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