Uncertainties in crystallization of hen‐egg white lysozyme: reproducibility issue

Abstract

The reproducibility of biomacromolecular crystallization (tetragonal and orthorhombic lysozyme crystals) was studied by monitoring the evolution of protein concentration during the crystallization process using Mach-Zehnder interferometer. It was found that formation of both tetragonal and orthorhombic crystals exhibited poor reproducibility. When the crystallization occurred under isothermal conditions, the protein concentration in the solution varied differently in different experiments under identical conditions (for both types of crystals). Moreover, in the case of orthorhombic lysozyme crystallization (under either isothermal or thermal gradient conditions), it is clear that the crystals could not be always readily formed. When formation of tetragonal lysozyme crystals was conducted at a temperature gradient condition, however, the evolution of concentration was reproducible. The phenomena found in this study revealed that biomacromolecular crystallization can be uncertain, which is probably caused by the process of nucleation. Such uncertainties will be harmful for the efforts of screening crystallization conditions for biomacromolecules.

Enhancement of nucleation during hanging drop protein crystallization using HF Treatment of cover glasses

“…If crystallization of a protein suffers from poor reproducibility, it will be difficult either to determine all of the crystallization conditions in the screening stage or to reproduce the crystallization that has been labeled as a "crystallizable" condition in the optimization stage. Therefore, improving reproducibility should be a big issue for practical protein crystallization [2,22].…”

“…Crystallization reproducibility It is known that protein crystallization often suffers from poor reproducibility, i.e., crystallization might or might not occur at identical crystallization conditions [2]. Therefore, testing a crystallization solution condition using only one experimental trial may be insufficient to determine whether crystals can form in that solution.…”

“…However, a normal Sparse Matrix Screen may not find all of the crystallization conditions in the kits due to problems in reproducibility [2]. Effort has been made to enhance the success rate of screens through improving the reproducibility of screens.…”

Surface treatment by oxidizing the plates can alter the response of protein crystallization

“…Crystallization reproducibility tests. To verify the crystallization results from the screening tests, we conducted reproducibility tests (Yin et al, 2008). Using the 96-well crystallization plates, we set up 96 repeated conditions.…”

Effect of the weather conditions during solution preparation on lysozyme crystallization

“…The difficulties may originate from the protein molecule itself if it is too flexible, too unstable or too large, or they may originate from unsuitable growth conditions if the crystallization recipes, concentrations, temperature, pH and other environmental factors are not appropriate (Rigaud et al, 2000;Samatey et al, 2000;Hui & Edwards, 2003). Because of these difficulties, protein crystallization often exhibits notoriously poor reproducibility: protein crystal growers often complain that the same crystallization conditions do not yield crystals again after the first successful crystallization (Yin et al, 2008). If a crystallization recipe has the potential to yield crystals, but not in a limited number of screening trials owing to poor reproducibility, this recipe will be ignored in the subsequent optimization process.…”