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

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.

Protein crystallization in short-peptide supramolecular hydrogels: a versatile strategy towards biotechnological composite materials

“…S1B †) and in the case of thaumatin crystals, the complete dissolution required 24 hours (Fig. This can be simply explained by the fact that protein crystals incorporate hydrogel fibers during their growth 11,14,37 and therefore, proteins have to diffuse through the protein-free hydrogel. As pointed out by Jones and Ulrich, protein crystals may be considered as solvates of salts, 34 which may explain the complex dissolution behaviour observed with the lysozyme polymorph.…”

“…[1][2][3] In most cases, the optimal strategy to obtain crystals of a particular protein is found serendipitously. This can be explained by (i) the physical properties of the hydrogel which eliminates sedimentation, convection current 7 and acts as impurity filter media, 8 and (ii) their molecular influence, as hydrogel fibers can interact directly with protein molecules, 9,10 having a final composite formed by polymeric fibers of agarose, 11 silica, 12,13 and PEG-based hydrogels, 14 incorporated within the crystal lattices of protein crystals. 5 It has been demonstrated that the use of conventional macromolecular hydrogels such as agarose, polyacrylamide, silica and sephadex has a direct impact on the formation of protein crystals and their quality.…”

The Finding of Nondissolving Lysozyme Crystals and Its Significance for the Study of Hard-to-Crystallize Biological Macromolecules

“…10 Some of the examples already adopted in structural genomic projects involve novel nucleants 11 and seeding policies, 12 or crystallization in gel media. 13 Past contributions from our group showed the beneficial effects on the formation and quality of protein crystals of ultrasonic irradiation, 14 small temperature oscillations, 15 and controlled supersaturation build-up profiles. 16 In turn, the biochemical approach actuates upon the phases that precede nucleation through the development of new ways to stabilize the macromolecular conformation.…”

Synthesis and characterization of a pH-responsive poly(ethylene glycol)-based hydrogel: acid degradation, equilibrium swelling, and absorption kinetic characteristics

“…PEG is a neutral, nontoxic, water-soluble polymer approved by the US Food and Drug Administration (FDA) for a variety of clinical uses, such as carrier and excipient in the pharmaceutical, cosmetic, and food products [5,11]. PEG has also been used in drug delivery, tissue engineering scaffolds, surface functionalization, and so forth [12,13]. Researchers in the area of biotechnology have focused on thermo-sensitive, pH-responsive, and (bio)degradable hydrogels that may be obtained from PEG [14].…”

“…and solid-state13 C-CP/MASThe 1 H-NMR spectra were recorded in a Varian spectrometer, model Mercury Plus BB 300 MHz, using a frequency of 300.059 MHz for 1 H nucleus, an angle pulse of 90°, and a relaxation delay of 30 s. The spectra were obtained with CDCl 3 solutions containing 20 mg L −1 of sample. The chemical shift was reported as δ values (ppm).…”