Utilisation of adsorption and desorption for simultaneously improving protein crystallisation success rate and crystal quality

Abstract

High-quality protein crystals of suitable size are an important prerequisite for applying X-ray crystallography to determine the 3-dimensional structure of proteins. However, it is often difficult to obtain protein crystals of appropriate size and quality because nucleation and growth processes can be unsuccessful. Here, we show that by adsorbing proteins onto porous polystyrene-divinylbenzene microspheres (SDB) floating on the surface of the crystallisation solution, a localised high supersaturation region at the surface of the microspheres and a low supersaturation region below the microspheres can coexist in a single solution. The crystals will easily nucleate in the region of high supersaturation, but when they grow to a certain size, they will sediment to the region of low supersaturation and continue to grow. In this way, the probability of crystallisation and crystal quality can be simultaneously increased in a single solution without changing other crystallisation parameters.

Snake Venom Peptides: Tools of Biodiscovery

“…We routinely perform crystallization experiments of peptides and proteins to apply crystallography in combination with highly intensive synchrotron radiation. In this context, we also developed new methods to improve protein crystallization [ 198 , 199 , 200 ]. Although realizing the highly flexible nature of some venom peptides and difficulties in crystallization, multidirectional NMR spectroscopy can be applied [ 201 ].…”

High Protein-Loading Silica Template for Heterogeneous Protein Crystallization

“…55 As the nucleants interacted favorably with the proteins, sorption took place, and the protein concentration in the droplets decreased. 56 There is limited literature on the use of high protein-loading nucleants for protein crystallization, especially about how the sorption of protein affects the effectiveness of the nucleant in accelerating crystallization. The current study aims to address this knowledge gap by using a mesoporous silica with characteristics similar to rod-like SBA-15 in the crystallization of lysozyme and thaumatin at the 1 mL scale.…”

“…Among these nucleants, various mesoporous silicas have been found to be effective nucleants for proteins. ,,− One promising candidate is rod-like SBA-15, which has been used for protein immobilization due to its high protein loading capacity. − The previous studies on heterogeneous nucleants were mostly conducted in the hanging-drop mode, where the nucleants were added directly into the protein/precipitant mixture droplets without any pretreatment . As the nucleants interacted favorably with the proteins, sorption took place, and the protein concentration in the droplets decreased …”

Comparison of the Quality of Protein Crystals Grown by CLPC Seeds Method

“…In this method, seed crystals are mainly inoculated into the pre-balanced protein crystal solution in metastable state, and the nucleation process is shortened by introducing foreign seed crystals to induce protein nucleation at low supersaturation, so as to obtain a highly ordered protein structure. To improve the quality of protein crystals by using tools such as electric fields [4,5], magnetic fields, ultrasound waves, light, mechanical vibration [6], microgravity [7,8], and nucleants [9], has always been studied. The addition of nucleants is one of the most commonly used methods for improving the quality of protein crystals.…”