Utilization of Cyclodextrins and Its Derivative Particles as Nucleants for Protein Crystallization

Abstract

Finding new nucleants to promote protein crystallization is an important task, especially for purposes other than structural determination. Here, we investigated cyclodextrins and its derivative particles, as potential nucleants for protein crystallization. β-Cyclodextrin (β-CD) and its derivatives (including p-toluenesulfonyl-β-cyclodextrin (PTCD), polymer-β-cyclodextrin (PCD), mono-(6-(1,6-hexamethylenediamine)-6-deoxy)-β-cyclodextrin (MHCD) and mercapto-β-cyclodextrin (MCD)) were used as nucleants. The experimental results confirmed that β-CD and its derivatives showed significantly positive effects, promoting protein crystallization and improving crystal quality. A larger number of protein molecules (including lysozyme, catalase, subtilisin A VIII, concanavalin A VI, α-chymotrypsinogen, proteinase K, cellulase, papain, glucose isomerase, hemoglobin, and ribonuclease A XII) attached to the particles usually corresponded to a higher crystallization success rate. More detailed analysis showed that cyclodextrins exhibited the best performance when the overall charge of protein in solution was the opposite to zeta potential of the cyclodextrins particle. Our results showed that cyclodextrins can be useful as nucleants due to the ease of modifying them to suit the crystallization of different proteins, and they can be explored for use in the mass purification of proteins for the biopharmaceutical industry. Furthermore, the phenomenon discovered in this study pointed toward a way to find new nucleants based on the overall charge of proteins in a solution: the nucleants should preferably be the opposite between the overall charge of target protein and the zeta potential of the cyclodextrin particle.

Cromoglycate mesogen forms isodesmic assemblies promoted by peptides and induces aggregation of a range of proteins

“… 39–41 Other non-denaturing chemicals such as cyclodextrins have also been used as nucleants for seeding protein aggregates and crystals. 42 For this consideration, we note that 5′DSCG also does not denature protein folding. 10 Furthermore, it appears that using PEG as a precipitant for protein crystallization requires a higher concentration range (∼5–45 wt%) 35–39 compared to 5′DSCG shown in this study (see below).…”

“…Chemical agents that can precipitate or aggregate proteins are critical for protein crystallization; [34][35][36][37][38][39][40][41][42] yet, there is no theoretical or empirical set of rules to guide the choice of precipitants in crystallizing a specic protein. For this reason, chemical screening kits have been developed to screen for large sets of reagents and conditions for protein crystallization.…”

Direct Crystallization of Proteins from Impure Sources

“…More recently, the sharp decrease in the requirement of protein solution volume (from several microliters to several nanoliters) enabled large scale crystallization screening with the help of modern automation techniques. − Moreover, hundreds of commercialized crystallization screening kits are available for crystallization screening, , providing us with tremendous opportunities that were unimaginable in the past. In addition to the screening methods, the implementation of many new methods to increase the nucleation, such as the utilization of numerous different nucleants, − microseeding, desiccation, , cross-diffusion microbatch (CDM), − and so on, largely increased the opportunities to determine the crystallization conditions.…”

Interface‐based crystal particle autoselection via membrane crystallization: From scaling to process control

“…Crystallization is one of the most important solid product separation and preparation technologies that have been widely applied in chemical, environmental, pharmaceutical, and food industries . Despite the maturity of this technology, the accurate and stable control of the crystallization process is still the fundamental issue limiting the reproducibility of product quality and process efficiency …”