Single-crystal – oriented-crystal – and epitaxy growth processes; – Apparatus – For crystallization from liquid or supercritical state
Reexamination Certificate
2001-03-30
2002-06-25
Hiteshew, Felisa (Department: 1765)
Single-crystal, oriented-crystal, and epitaxy growth processes;
Apparatus
For crystallization from liquid or supercritical state
C117S200000, C117S900000, C422S245100
Reexamination Certificate
active
06409832
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to a device for growing crystals, and, more particularly, to a device for promoting protein crystal growth using microfluidic structures.
2. Description of the Related Art
Macromolecular crystals have become keystones of molecular biology, biochemistry, and biotechnology. Understanding how crystals express their function depends on knowledge of the macromolecular architecture at the atomic level.
The determination of the three dimensional atomic structure of crystals is one of the most important areas of pure and applied research. This field, known as X-ray crystallography, utilizes the diffraction of X-rays from crystals in order to determine the precise arrangement of atoms within the crystal. The result may reveal the atomic structure of substances as varied as metal alloys to the structure of deoxyribonucleic acid (DNA). The limiting step in all of these areas of research involves the growth of a suitable crystalline sample.
One important and rapidly growing field of crystallography is protein crystallography. Proteins are polymers of amino acids and contain thousands of atoms in each molecule. Considering that there are 20 essential amino acids in nature, one can see that there exists virtually an inexhaustible number of combinations of amino acids to form protein molecules. Inherent in the amino acid sequence or primary structure is the information necessary to predict the three dimensional structure. Unfortunately, science has not yet progressed to the level where this information can be obtained quickly and easily. Although considerable advances are being made in the area of high field nuclear magnetic resonance, at the present time the only method capable of producing a highly accurate three dimensional structure of a protein is by the application of X-ray crystallography. This requires the growth of reasonably ordered protein crystals (crystals which diffract X-rays to at least 3.0 angstroms resolution or less), as the accuracy of structures determined by X-ray crystallography is limited by the disorder in the crystallized protein.
The maximum extent of a diffraction pattern is generally considered to be a function of the inherent statistical disorder of the molecules of protein crystals rather than the result of purely thermal effects. Statistical disorder present in protein crystals has two principal sources: 1) intrinsic structural or conformational variability of protein molecules, and 2) spatial distribution of the individual molecules about lattice sites occupied.
In addition, other inherent limitations in the crystallization process involve the effects of molecular convection, thermal effects, and buoyancy, all due to the earth's gravitational field. Therefore, it has been proposed to conduct crystallization experiments in the microgravity ({fraction (1/1000)} g to {fraction (1/10,000)} g) of space, on board the space shuttle, international space station, or other similar vehicles. Several patents disclose crystallization in microgravity to improve the size, morphology and diffraction quality of crystals. U.S. Pat. Nos. 5,362,325 and 4,755,363 are exemplary of patents disclosing microgravity crystallization.
Focus of microgravity research in protein crystal growth (PCG) has been based on the observation that PCG in a microgravity environment yields protein crystals that are of reduced disorder. Reduction in lattice disorder by protein crystals grown in microgravity compared to ground controls offers enhanced resolution of diffracted intensities and translates at the atomic level into more precise knowledge of the protein architecture. The detailed knowledge of how ligands interact with binding sites at the atomic level permits insight into catalytic mechanisms and recognition in biological systems, a prerequisite for structure-assisted drug design. In a pharmaceutical industry setting, higher resolution implies significant manpower reduction in synthetic chemistry to explore the drug-binding site and results in more rapid optimization of drug target interaction. Accelerated drug design is extremely cost effective, allowing a pharmaceutical company to quickly recover R&D costs and improve profitability.
Several important advances have recently accelerated the structure determination process using even small crystals. These include selenomethionyl proteins, cryo-crystallography, high intensity synchrotron radiation sources, CCD detectors, and multiwavelength anomalous diffraction (MAD) phasing. With these advances, a protein structure can be solved by MAD phasing literally within hours of data collection at a synchrotron radiation source. The outstanding uncertainty faced by protein crystallography is the growth of high quality protein crystals.
In the very near future, it is expected that the field of structural genomics will foster a tremendous explosion in demand for protein structure determination. Genome sequencing or genomics is significantly impacting biological research by changing our understanding of biological processes through identification of novel proteins that may be involved in disease or are unique to pathogenic organisms. Genome project results have shown that in most organisms, more than 50% of the proteins have no assigned function. In the human genome, this amounts to over 50,000 proteins. These uncharacterized proteins thus represent a reservoir of untapped biological information that is widely acknowledged as the next generation of protein therapeutics and targets for pharmaceutical development. With large-scale genomic sequencing now becoming routine, attention is being focused on understanding the structure and function of these biological macromolecules. Recently published examples where knowledge of a three-dimensional structure of an unknown protein can provide clues to its function is expected to open the gates to a massive need for high quality structure determination.
The crystallization process generally involves several distinct phases, such as nucleation and post-nucleation growth. Nucleation is the initial formation of an ordered grouping of a few protein molecules, while post-nucleation growth consists of the addition of protein molecules to the growing faces of the crystal lattice and requires lower concentrations than the nucleation phase.
Most protein crystals nucleate at very high levels of supersaturation, typically reaching up to 1000% in many cases. At such supersaturation levels, post-nucleation crystal growth takes place under very unfavorable conditions. Most macromolecules at the concentrations needed to attain the very high levels of supersaturation tend to form aggregates and clusters of both ordered oligomeric species and/or random amorphous aggregates. Depending on the half-life and concentration of such clusters, formation of nuclei can involve incorporation of partially ordered aggregated species. Quiescent conditions mitigate imperfect post-nucleation growth at high supersaturation by reducing the collision frequency of aggregate species of all kinds to form larger clusters or nuclei. Microseeding a protein solution, that is, introduction of freshly crushed crystallites, would provide a succinct approach to circumvent growth from imperfect nuclei.
At higher levels of supersaturation, growth by absorption of three-dimensional nuclei onto crystal faces has been observed in crystallization studies of thaumatin, catalase, t-RNA, lysozyme, lipase, STMV virus and canavalin. The three-dimensional nuclei have observed average dimensions ranging between 1-10 &mgr;m making them colloidal in size. The origin of these nuclei is thought to be protein clusters that originate from protein rich droplets possessing short-range internal order and that undergo long-range ordering upon interaction with the underlying crystal lattice.
Under quiescent conditions at low supersaturation, a protein crystal grows by incorporation of individual protein molecules, monomers, from the surrounding medium, which because of their low diffusivities p
Sygusch Jurgen
Weigl Bernhard H.
Hiteshew Felisa
Litzinger Jerrold J.
Micronics, Inc.
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