Tuning Transport Phenomena in Agarose Gels for the Control of Protein Nucleation Density and Crystal Form Artusio, Fiora Castellví, Albert Pisano, Roberto Gavira Gallardo, José Antonio Protein Crystallization Polymorphism Agarose gel Nucleation This research was funded by the project BIO2016-74875-P (MINECO), Spain, co-funded by the Fondo Europeo de Desarrollo Regional (FEDER funds), European Union. We are very grateful to the Spanish Synchrotron Radiation Facility (ALBA) for their provision of synchrotron radiation facilities, and we would like to thank the XALOC staff for their assistance and support during the data collection. 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, P2(1)2(1)2(1), was obtained, and the three-dimensional structure was determined at a 1.6 angstrom 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. 2021-06-11T10:30:24Z 2021-06-11T10:30:24Z 2021-04-22 info:eu-repo/semantics/article Artusio, F.; Castellví, A.; Pisano, R.; Gavira, J.A. Tuning Transport Phenomena in Agarose Gels for the Control of Protein Nucleation Density and Crystal Form. Crystals 2021, 11, 466. [https://doi.org/10.3390/cryst11050466] http://hdl.handle.net/10481/69129 10.3390/cryst11050466 eng http://creativecommons.org/licenses/by/3.0/es/ info:eu-repo/semantics/openAccess Atribución 3.0 España MDPI