Abstract: The temporal and spatial growth behaviour of protein crystals, subject to different cooling strategies in protein crystallisation was investigated. Although the impact of temperature and cooling rate on crystal growth of small moleculeswaswell documented, much less has been reported on their impact on the crystallisation of proteins. In this paper, an experimental set-up is configured to carry out such a study which involves an automatic temperature controlled hot-stage crystalliser fittedwith a real-time imaging system. Linbro parallel crystallisation experiments (24-well plate)were also conducted to find the suitable initial conditions to be used in the hot-stage crystallisation experiments, including the initial concentration of HEWlysozyme solutions, precipitate concentration and pH value. It was observed that fast cooling rates at the early stage led to precipitates while slow cooling rates produced crystal nuclei, and very slowcooling rates,much smaller than for smallmolecules are critical to the growth of the nuclei and the crystals to a desired shape. The interesting results provide valuable insight aswell as experimental proof of the feasibility and effectiveness of cooling as a means for achieving controlled protein crystallisation, compared with the evaporation approach which was widely used to grow single large crystals for X-ray diffraction study. Since cooling rate control can be easily achieved and has good repeatability, it suggests that large-scale production of protein crystals can be effectively achieved by manipulating cooling rates.

Key words: Hot-stage reactor, On-line imaging of crystal growth, Hen-Egg-White lysozyme cooling, crystallisation, Protein crystallisation, Real-time in-process imaging

摘要： The temporal and spatial growth behaviour of protein crystals, subject to different cooling strategies in protein crystallisation was investigated. Although the impact of temperature and cooling rate on crystal growth of small moleculeswaswell documented, much less has been reported on their impact on the crystallisation of proteins. In this paper, an experimental set-up is configured to carry out such a study which involves an automatic temperature controlled hot-stage crystalliser fittedwith a real-time imaging system. Linbro parallel crystallisation experiments (24-well plate)were also conducted to find the suitable initial conditions to be used in the hot-stage crystallisation experiments, including the initial concentration of HEWlysozyme solutions, precipitate concentration and pH value. It was observed that fast cooling rates at the early stage led to precipitates while slow cooling rates produced crystal nuclei, and very slowcooling rates,much smaller than for smallmolecules are critical to the growth of the nuclei and the crystals to a desired shape. The interesting results provide valuable insight aswell as experimental proof of the feasibility and effectiveness of cooling as a means for achieving controlled protein crystallisation, compared with the evaporation approach which was widely used to grow single large crystals for X-ray diffraction study. Since cooling rate control can be easily achieved and has good repeatability, it suggests that large-scale production of protein crystals can be effectively achieved by manipulating cooling rates.

关键词: Hot-stage reactor, On-line imaging of crystal growth, Hen-Egg-White lysozyme cooling, crystallisation, Protein crystallisation, Real-time in-process imaging