Batch crystallisation of lysozyme and catalase with the aid of silica nucleants

Abstract

There is a great interest in the use of heterogeneous seeds on the crystallisation of proteins, with most studies on the ability to obtain diffractive quality crystals in vapour diffusion. Reports by Shah et al (2011) developed a relationship between surface pore diameters of these seeds and the size of proteins for effective protein crystallisation. When there is a match between these properties, a significant reduction in induction time can be achieved with vapour diffusion experiments. Heterogeneous seeds of such size match are named ‘nanotemplates’ by Shah and co-workers. This thesis aims to further understand the effects of the porous properties on the nucleation behaviour of proteins. Specifically, the effects in batch crystallisation will be studied, in which both the thermodynamics and kinetic pathways are expected to be significantly different. In order to achieve this, the induction time was used as a property to represent the nucleation process for proteins and was monitored using both UV-vis spectroscopy and turbidimetry. Hen Egg White Lysozyme (HEWL) and Bovine Liver Catalase (BLC) are used as model proteins for the studies. Mesoporous heterogeneous seeds used are of pore diameters 5.5 and 9.0, and non-porous seeds are also used for comparison. These seeds would be represented as NT40, NT120 and NP respectively throughout the thesis. The protein hydrodynamic diameter and the pore diameters were characterised by Dynamic Light Scattering and Nitrogen Adsorption techniques respectively prior to crystallisation studies. For studies involving HEWL, batch crystallisation was applied directly due to the vast details of thermodynamic behaviour available. Studies with lysozyme also serve as a purpose to establish a basic understanding of different parameters (seed concentration, seed type, supersaturation, stirring), which can be applied to the crystallisation of BLC. However, studies regarding BLC required the determination of solubility prior to the studies involving the use of seeds, and experiments were demonstrated at different volumes. In these studies, different seed concentrations were investigated to a) identify a suitable seed concentration range for heterogeneous seeding and b) investigate the varying amount of surface properties (pore diameter, surface area). Different protein concentrations (13.5 – 17.5 mg/mL) was also compared due as heterogeneous surfaces are expected to have different extent of contribution. For the crystallisation of HEWL, the use of seed reduced the induction time compared to unseeded experiments. Unexpected results were obtained where NT40, the seed with pores iv of closest match to the protein, resulted in the longest induction time across all seed concentration levels tested (0.05 -2.00 mg/mL), and NP resulted in the shortest induction time, especially at low seed concentrations (0.05 and 0.1 mg/mL). NT120 gave an intermediate result between the two. For all seed types used, it appears that the induction time is constant at certain ranges of seed concentration, and the values for these ranges varies for different seed types. In other words, the induction time dependence on amount of seeds (hence amount of surface properties and pore volumes) is not linear for the seed concentration ranges tested. As expected, the effects of heterogeneous seeds are more significant at reduced protein concentrations. At 13.5 mg/mL, the induction time for lysozyme was over 10 hours when unseeded, the use of nanotemplates (1.25 mg/mL) reduced the induction time by ~75%, and the use of other seeds at the same seed concentration reduces the induction time to by ~80 %. Conditions in which crystallisation of BLC occurs was identified. As there are limited information available in literature on the solubility of catalase, this property was determined for the protein at 20 °C, in solution conditions of precipitant concentration 8-10% PEG 4000, 5 % MPD in KPO4 (50 mM) buffer at pH 7, in which crystallisation experiments were conducted for this protein, and was found to be ~ 10.5 mg/mL. Based on this, crystallisation was conducted at 13.4 mg/mL and 17.5 mg/mL at 8 % PEG (m/v), which corresponded to supersaturation ratios 1.28 and 1.67 respectively. Stirred batch crystallisation experiment was conducted at 0.5 mL and 20 mL scale. At 0.5 mL, even at low stirring, nucleation occurred instantaneously at a seed concentration of 1 mg/mL. At 20 mL working volume and a stirring rate of 100 rpm, there was a slight increase in induction time compared to the 0.5 mL experiments. At this scale, experiments were conducted at BLC concentrations at 17.5 mg/mL and 13.4 mg/mL, and a range of seed concentrations were investigated. Non-porous material results in a slight decrease in induction time at 17.5 mg/mL, while at 13.4 mg/mL, the effects of NT 120 were more prominent at high seed concentration (>2.0 mg/mL). Demonstrating the different surface property contributes differently to the influences on crystallisation induction time of protein at different supersaturations. It is shown that batch crystallisation of proteins at batch scale (~20 mL) is achievable, and that the use of heterogeneous nucleants assisted in the nucleation/crystallisation of proteins. Results presented in this thesis highlights the relationship between nucleant concentrations used and its effect on induction time, and also the supersaturation dependency of the effects of nanopores compared to non-porous surfaces.