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

Crystals

Fiora Artusio¹  Roberto Pisano¹  JoséA. Gavira²  Albert Castellví³ 

[1] Department of Applied Science and Technology, Politecnico di Torino, 24 Corso Duca degli Abruzzi, 10129 Torino, Italy;Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra (Consejo Superior de Investigaciones Científicas-Universidad de Granada), Avenida de las Palmeras 4, 18100 Armilla, Spain;Molecular Biology Institute of Barcelona, Carrer Baldiri Reixac 4-8 (Science Park), 08028 Barcelona, Spain;

关键词: protein;    crystallization;    polymorphism;    agarose gel;    nucleation;   

DOI  :  10.3390/cryst11050466

来源: DOAJ

【 摘 要 】

Agarose gels provide the ideal environment for studying the nucleation step of complex biomacromolecules under diffusion-controlled conditions. In the present paper, we characterized the influence of agarose on the nucleation of three model proteins, i.e., lysozyme, insulin, and proteinase K, as a function of the agarose concentration using a batch method set-up inside flat capillaries. By using this set-up, we were able to directly count the number of crystals in a given volume and correlate it with the amount of agarose and with the average crystal size. We also studied the crystallization behavior of proteinase K with free-interface diffusion so that batch conditions were achieved through slow diffusion of the precipitant. Thanks to the control over the protein mass transport imposed by the network, a previously unknown crystal form, P2₁2₁2₁, was obtained, and the three-dimensional structure was determined at a 1.6 Å resolution. Overall, the versatility of agarose gels makes them ideal candidates for the preparation of microcrystalline suspensions of biopharmaceuticals with precise and reproducible crystal attributes or for the exploration of the existence of different polymorphs.

【 授权许可】

Unknown