Project: Innovative protein crystallization technology for structure determination of drug targets.

Three-dimensional (3-D) structural information of protein drug targets facilitates saving time and reducing costs of drug development. Structural Biology aims to determine the 3-D atomic structure of proteins, giving insight into the function of these molecules in the living cells; X-ray crystallography of protein crystals being the method mostly used. However, protein crystallization, especially of insoluble membrane proteins, creates a severe limiting step in the complex task of structure determination._x000D__x000D_The proposed project will combine the revolutionary innovative protein crystallization technique of CrystalClear Pship with leading organizations for Structure Biology in Europe: The Grenoble Outstation of the European Molecular Biology Laboratory (EMBL) and the European Synchrotron Radiation Facility (ESRF), both located in Grenoble, France. Together, we aim to overcome the limitations of current protein crystallography techniques, and set up a unique service for protein structure determination to the benefit of scientific research and structure based drug discovery. _x000D__x000D_In the structure determination pipeline, proteins have to be cloned, expressed, purified crystallized and then subjected to diffraction experiment. The resulting crystals have to be of quality and size to provide a high resolution diffraction pattern at a modern X-ray source (typically a synchrotron). Analysis of the diffraction data allows the construction of a three-dimensional model of the molecule. Crystallization is one of the major bottlenecks in this process. Standard methods for protein crystallization rely on the addition of precipitants to a highly concentrated protein solution. Vapor diffusion techniques are then used to produce a supersaturated solution from which crystals will grow. The most popular experimental set ups are based on hanging drop or sitting drop configurations. In both cases, a droplet containing the protein sample is mixed with a droplet of precipitant solution and allowed to equilibrate in a closed system (reservoir) with a large volume of the same precipitant solution at higher concentration. As water diffuses from the drop into the reservoir, both protein and precipitant concentrations in the drop increase and as over-saturation occurs, crystallization may take place there. However, more often, the result of this process is an amorphous precipitate. Hence it is necessary screen wide variety of precipitants under different conditions, requiring large amounts of sample and time. This makes the process of protein crystallization costly, and its success rate is rather low, especially for proteins that reside in cell membrane which are important drug targets. In reality, the success rate in crystallization of proteins is about 35%, falling to 10% for membrane proteins. In recent years, high cost robotic systems have been developed, offering high throughput approaches. Nevertheless, crystallography continues to be a major limiting step in protein structure determination. _x000D__x000D_The new crystallization technology proposed to be further developed in this project, replaces the standard unsatisfactory crystallography techniques by applying isoelectric focusing as a means of concentrating proteins in a continuous isoelectric gel medium. The immediate benefits resulting from this technology in comparison to those being currently used are: elimination of the screening process, relaxed purity requirements, lower initial protein concentration, a much shorter timescale (the time to crystallization brought down from weeks or months to a matter of hours), increased throughput, lower costs, and the most significant breakthrough - the ability to crystallize hydrophobic or low-solubility proteins including those of membrane origin. _x000D__x000D_During the proof of concept phase experiments, tens of proteins (including integral membrane proteins) were crystallized using CrystalClear's innovative method yielding a success rate of 100%. Having only limited access to proper X-ray facility at the time, quality tests for structure determination adequacy were done on a small number of crystals produced. Those produced high resolution (1.7 A ) diffraction data._x000D__x000D_The proposed joint program will continue the development of CrystalClear's technology by standardizing the crystallization platform and by improving crystals harvesting and transfer to the X-ray beam-lines. The program includes also prototyping of a commercial crystallizing device. The final outcome of the project will be a unique, commercial joint ESRF-EMBL-CrystalClear protein crystallography service for structure determination._x000D_

Acronym CrystalClear (Reference Number: 4280)
Duration 03/06/2009 - 01/07/2011
Project Topic The proposed project will validate a novel and proprietary technology platform of crystallization for protein structure determination, avoiding CO limits of existing methods and enhancing drug discovery and basic protein science. Commercial protein structure analysis service will be established.
Project Results
(after finalisation)
A new crystallization method has been developed and validated with a number of model proteins. This method could potentially be applied to the structural analysis of difficult to crystallize proteins which are potential targets for drug development. Robust standard operation protocols are to be further developed before commercial exploitation.
Network Eurostars
Call Eurostars Cut-Off 1

Project partner

Number Name Role Country
3 European Synchrotron Radiation Facility Partner France
3 CrystalClear Pship Coordinator Israel
3 European Molecular Biology laboratory, Grenoble Outstation Partner France