High Protein-Loading Silica Template for Heterogeneous Protein Crystallization

Abstract

As a purification technology, crystallization is advantageous over chromatography and precipitation in terms of purity, cost, and scalability. In general, proteins have slow crystallization kinetics due to their complex configurations, but this problem can be overcome with heterogeneous nucleants. High protein-loading mesoporous silica is a promising nucleant due to its favorable interaction with protein. The current study employs such a template in the batch crystallization of lysozyme and thaumatin at 1 mL scale. Using lysozyme as the main model protein, the results show that the template had high protein loading (220–300 mg lysozyme/g silica) and induced significantly faster crystallization as compared to the unseeded samples over a wide range of initial protein concentration (10.0–47.5 mg/mL) at 25 °C with 0.5 M potassium sodium tartrate tetrahydrate (precipitant) concentration. It was found that the template had to be saturated with the target protein before the experiments to achieve faster kinetics, or else it could delay the crystallization process. These findings were reaffirmed by the crystallization experiments of thaumatin. The current study demonstrates the effectiveness of high protein-loading silica as a nucleant in protein crystallization and the importance of its pretreatment with the target protein.

Theoretical and experimental investigation of protein crystal nucleation in pores and crevices

“…The plausible explanation for HAP acting as an antinucleant in the case of lysozyme seems to be that HAP itself lowers the protein concentration in the system via sorption (Chen et al, 2020), thus making the solution concentration too close to equilibrium. This anti-nucleant effect can be useful in cases where there is excessive nucleation.…”

Protein crystallisation facilitated by silica particles to compensate for the adverse impact from protein impurities

“…34 Researchers also showed that porous silica have positive impact as heterogenous nucleants. 32,33,35 As shown in Table 1, mesoporous silica had the highest surface area while non-porous silica had the lowest. Contradicting our hypothesis, non-porous silica worked better on improving lysozyme crystallisation comparing to mesoporous silica.…”

“…However, some limited cases of selective crystallisation were also described. 26,[32][33][34][35] Nonetheless, systematic knowledge of crystallisation behaviour of target protein from the mixture is still absent. The reported selective crystallisation cases only focused on specific scenarios either with ultralow protein concentration, with a very limited range of protein composition of the mixture, or at relatively small scale and long-time span which are not suitable for industrial application.…”

Enhancing the crystallisation of insulin using amino acids as soft-templates to control nucleation

“…The interplay of these effects results in a significantly faster crystallisation and reduces the necessary bulk-supersaturation needed to form nuclei (15,17,18). Heterogeneous nucleants include mineral surfaces (19), membranes (20), polymers (21), DNA-origami (22), nano-and mesoporous particles (23,24), porous surfaces (25,26), and surfaces with selected functional groups (4,17,25,27). Selective nucleants that have an engineering surface topography and morphology, such as molecular imprinted silica surfaces or polymers, have the ability to selectively crystallise proteins due to favourable geometrical and physico-chemical interaction between the protein and the nucleant's surface (4,15,18,28,29).…”