Single-crystal – oriented-crystal – and epitaxy growth processes; – Apparatus – With means for measuring – testing – or sensing
Reexamination Certificate
2000-12-12
2003-07-22
Kunemund, Robert (Department: 1765)
Single-crystal, oriented-crystal, and epitaxy growth processes;
Apparatus
With means for measuring, testing, or sensing
C117S068000, C117S069000, C117S901000
Reexamination Certificate
active
06596081
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the formation of crystals of small and large molecules, including macromolecules such as proteins and nucleic acids, and more particularly, to a method and apparatus for dynamically controlling the process of forming such crystals, and the resulting crystals.
2. Description of the Related Art
There is great interest in determining the three-dimensional (3-D) structures of biological molecules. Ongoing studies of the genomes of humans and other mammals, as well as of disease-causing viruses, bacteria, and parasites, are identifying thousands of genes and proteins, many of which are linked to diseases of humans and domestic animals and plants. Knowing the 3-D structures of the molecules that are involved in causing a disease can be a tremendous aid in developing drugs to prevent or treat the disease. Rational drug design involves obtaining a precise 3-D structure of a specific molecule involved in a disease process, making and studying physical models and computer-generated graphic images of the structure, as well as using sophisticated computer programs that thermodynamically model the structure and its interactions with solvent and other molecules, in order to design drugs that selectively bind to and alter the function of the disease-causing molecule. Rational drug design can be used to analyze and design drugs that interact with small molecules such as peptide and non-peptide hormones, as well as intermediate-sized and large macromolecules such as nucleic acids and proteins, respectively. The application of rational drug design will result in a number of diseases and pathological symptoms being brought under control. For example, CAPTOPRIL is a well known drug for controlling hypertension that was developed through rational drug design techniques. CAPTOPRIL inhibits generation of the angiotension-converting enzyme thereby preventing the constriction of blood vessels.
X-ray crystallography is a technology that allows us to obtain the precise 3-D atomic structures of molecules such as peptides, proteins, and nucleic acids. A critical step in using X-ray crystallography to determine the 3-D structure of any molecule of interest is establishing a reliable method for crystallizing the molecule. Proteins are one of the major classes of structural molecules in living organisms; protein enzymes catalyze the metabolic reactions that make life possible; and many disease processes are mediated by the interactions of proteins with other molecules, including other proteins. Therefore, much time and money have been spent crystallizing proteins for analysis of their structures. Recent biotechnological developments in cloning and over-expression of genes encoding proteins of interest and in the purification of proteins are increasing the need for a reliable way in which to grow protein crystals. Unfortunately protein crystallization is a difficult and unpredictable art.
Crystallization of a biological molecule such as a protein involves the creation of a supersaturated solution of the molecule under conditions that promote minimum solubility and the orderly transition of the molecules from the solution into a crystal lattice. The variables that must be controlled precisely to promote crystal growth include temperature, protein solution concentration, salt solution concentration, pH, and gravitational field, for example (Durbin, S. & Feher, G. Ann Rev Phys Chem 47 (1996) 171-204, the entire contents of which are incorporated herein by reference). These variables are carefully controlled and optimum combinations thereof are determined through experimentation to yield superior crystals.
In the description of the invention that follows, the molecule being crystallized, e.g., the protein, is referred to as a “reactant,” and the solution containing one or more precipitants that is used in crystallization processes is referred to as a “reagent” solution.
The crystallization process generally involves three distinct phases; nucleation, sustained crystal growth, and termination of crystal growth. Nucleation is the initial formation of an ordered grouping of a few reactant molecules and requires a particular concentration of reactant molecules in a precipitating reagent solution. On the other hand, the continued growth phase consists of the addition of reactant molecules to the growing faces of the crystal lattice and requires lower concentrations of reagent solution than the nucleation phase. The termination phase can be initiated by poisoning the growing lattice with denatured or chemically modified reactant molecules or with different molecules, by depletion of the reactant solution, or by changing the concentration of precipitant to a specified level.
It is considered desirable to obtain a small number of crystallization nuclei quickly that will grow slowly into full-sized crystals. Theoretically, this allows for a relatively large size of the resulting crystals, homogenous crystal order and morphology, and balanced crystal dimensions. Therefore, it is desirable to begin crystallization with a reagent solution containing a particular concentration of precipitant until nucleation is detected, at which point it is desirable to adjust the concentration of precipitant. Thus, one of the critical requirements of any molecular crystallization process is the fine and dynamic control of the various parameters that determine the concentration of the precipitant in the solution in which the target reactant molecule protein is suspended. This control requires the ability to attain nucleation conditions and the ability to modify the concentration of the precipitant without disturbing the crystallization process.
There are several conventional techniques for forming molecular crystals; for example, liquid diffusion, vapor diffusion and dialysis techniques. These processes are relatively slow and cannot be readily controlled dynamically. Therefore, these processes require complex and large apparatus in order to control crystallization, if crystallization can be controlled at all. Accordingly, it is desirable to overcome these limitations.
Most conventional protein crystallization methods mix a solution containing the reactant protein molecules with a crystallizing, or precipitating, solution to accomplish crystallization. In terms of mechanics, use is commonly made of syringes, stepping motors, valves of various types, membranes to separate solutions, and in one case, a gel to replace the membrane and act as a delaying filter device between solutions. U.S. Pat. Nos. 4,917,707, 5,106,592, and 5,641,681, and Microdialysis Crystallization Chamber, L. C. Sieker, J. Crystal Growth 90 (1988) 349-357, the entire contents of which are incorporated herein by reference, disclose these concepts.
It is known to “control” the crystallization process. However, only the movement of liquids via pumps, valves and syringes is controlled in conventional crystallization processes. This control creates a static condition (bath concentration) which is predefined for the protein in question. For example, U.S. Pat. No. 4,755,363, the entire contents of which are incorporated herein by reference, discloses delivering liquids at desired flow rates and concentrations. However, U.S. Pat. No. 4,755,363 fails to disclose changing conditions within the crystallization chamber (with the exception of temperature) once those conditions have been set and crystallization has begun.
Temperature is an important parameter that can be controlled to optimize conditions separately for nucleation or growth. U.S. Pat. Nos. 4,755,363 and 5,362,325, the entire contents of which are incorporated herein by reference, are exemplary of patents disclosing temperature control in crystallization processes. U.S. Pat. No. 5,362,325 discloses varying the concentration of a crystallizing agent over time to produce a predetermined gradient in the concentration of the crystallizing agent. However, this reference fails to disclose dynamic control as disclosed for this invention.
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Arnowitz Leonard
Steinberg Emanuel
BSI Proteomics Corporation
Kunemund Robert
Spiegel H. Jay
LandOfFree
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