Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2

Abstract

The automated microbatch technique developed at Imperial College has been used to establish a phase diagram for crystallization. The concentrations of the protein (carboxypeptidase G2) and precipitant (PEG 4000) were varied, while pH and temperature were kept constant. The diagram consists of an undersaturation and a supersaturation zone, the latter being subdivided into the metastable, nucleation and precipitation zones. In the metastable zone, crystals may grow but nucleation of crystals does not occur. It is the best zone for growth of X.-ray diffraction quality crystals because of the slower growth rate and the avoidance of uncontrolled nucleation, which uses up protein in the formation of tiny crystals. Nevertheless, in practice, it is rarely well defined or used because nuclei must be introduced artificially into the system. The new method used here consists of setting crystallization droplets at nucleation conditions and later diluting them to conditions where nucleation has not been observed. Single diffracting crystals of typical dimensions 0.3 × 0.3 x 0.2 mm were routinely obtained in the metastable zone, equivalent to the best (very rarely) obtained crystals in the nucleation zone.

Use of Dual Polarization Interferometry as a Diagnostic Tool for Protein Crystallization

“…This discrepancy could be expected due to the "shock nucleation" effect in microbatch at high protein concentrations, when nuclei form at the interface where the two highly concentrated protein and precipitant solutions come into contact. Shock nucleation leads to an underestimation of the salt concentrations needed for protein precipitation 15 .…”

Trends and Challenges in Experimental Macromolecular Crystallography

“…Crystallization by means of variation of temperature and application of temperature gradients have successfully been applied to protein crystallization on the ground (e.g. Feigelson & DeMattei, 1992;Rosenberger et al 1993;Blow et al 1994) and also in microgravity (Long et al 1995). The problem remains, unfortunately, that not many proteins are sensitive to a temperature change.…”

“…This was confirmed by Hirschler et al (1995). The nucleation and consequently the number and size of lysozyme and of carboxypeptidase G 2 crystals was controlled at will by the addition of different quantities of a nucleant to filtered trials containing these proteins (Chayen et al 1993;Blow et al 1994).…”

Systematic investigation of protein phase behavior with a microfluidic formulator

“…There are a variety of schemes that manipulate the kinetics of the crystallization process, and all take advantage of generic features of these phase diagrams (4). However, in practice, the phase behavior of very few proteins has been studied in detail (5-12), and solubility information for a specific protein is rarely available for crystallization and optimization experiments (13,14).Furthermore, it is often an arduous process to find the right combination of chemicals that yields appropriate phase behavior for a given protein. Every protein is different, and even a modest subset of stock precipitating solutions comprises a vast chemical phase space that must be explored.…”

“…There are a variety of schemes that manipulate the kinetics of the crystallization process, and all take advantage of generic features of these phase diagrams (4). However, in practice, the phase behavior of very few proteins has been studied in detail (5-12), and solubility information for a specific protein is rarely available for crystallization and optimization experiments (13,14).…”