Agarose Gel as a Medium for Growing and Tailoring Protein Crystals

Abstract

The nucleation inducing ability of agarose gels has been exploited to study the crystallization of proteins in diffusion-dominated environments. The crystal size was successfully tuned in a wide range of gel, protein, and precipitant concentrations. The impact of the gel content on crystal size was independent of the specific protein, allowing the mathematical prediction of crystal size and pointing out the exclusivity of physical interactions between the gel and the protein. The versatility of the technique and the fine-tuning of the nucleation flux was demonstrated by crystallizing five different proteins and implementing batch and counter-diffusion crystallization. In addition, the potential of agarose gel to be used not only as a growth but also as a delivery medium for serial crystallography applications has been proven by preparing unidimensional microcrystal slurries with 0.1% (w/v) gel.

Tuning Transport Phenomena in Agarose Gels for the Control of Protein Nucleation Density and Crystal Form

“…Agarose gels allow the study of protein crystallization in diffusive environments thanks to the suppression of the convective mass and heat transport [36]. In our previous publication [25], a correlation between the protein crystal size measured at equilibrium and the agarose concentration was identified and was found to be independent of the protein's nature. All the evidence pointed out the ability of agarose gels to induce crystallization due to the physical interaction with the protein.…”

“…Agarose provides a neutral support for nucleation in terms of both net charge and chemical interaction with the proteins. In our previous publication [25], we employed agarose gels to grow protein crystals with tailored sizes depending on the agarose concentration. The inducing action of the gel on the nucleation processes was attributed to the favorable physical interactions with the crystallization environment.…”

Advanced Biocrystallogenesis

“…This method is effective due to the invariable environment enabling crystal growth, preventing the production of protein aggregates or sedimentation and slows diffusion of molecules in the crystallization chamber [13]. The most used gels for macromolecular crystallization are hydrophilic gels, namely agarose and silica gels, even though they have different properties triggering the crystal growth [14].…”

“…Regarding nucleation, agarose gels significantly promote it, whereas, silica gels inhibit this process, but the exact mechanism behind it remains unknown. The benefits of the use of agarose gels are (i) the concentration of agarose gel can be very low, (ii) agarose gel polymerises faster than silica gels, (iii) agarose gel can be employed in any crystallization technique, (iv) cofactors, ligands and even cryoprotectants are reported to be easily soaked into the crystal in the presence of the agarose gel, (v) agarose gels help to produce bigger crystals for neutron diffraction analysis and also (vi) serves as delivery medium for nanocrystals for X-ray freeelectron laser analysis (Figure 1-(3, 4)) [14][15][16][17].…”

Role of Self-Assembled Surface Functionalization on Nucleation Kinetics and Oriented Crystallization of a Small-Molecule Drug: Batch and Thin-Film Growth of Aspirin as a Case Study

“… 6 , 7 The latter relies on confinement to boost nucleation kinetics of APIs with polymeric gels 8 , 9 or promote protein crystallization with the help of agarose gels and mesoporous structures. 10 , 11 However, little attention has been paid to the deconvolution of topographical and chemical effects induced by surfaces on nucleation, often leading to the difficult interpretation of results. The isolation of the two components is desirable to rigorously understand the role of surface–API interactions during crystallization, and it may be achieved with smooth surfaces exposing different chemical groups.…”