Apparatus for crystal growth of biomacromolecules

Abstract

A method comprising the steps of continuously changing the concentrations in solution of a biomacromolecule to be crystallized and a precipitant, thereby constructing a crystal phase diagram containing a solubility curve, searching for optimum conditions of crystallization on the basis of the constructed crystal phase diagram, and performing efficient growth of the crystal of the biomacromolecule. Also disclosed is an apparatus for implementing the method.

What is claimed is:  
     
         1 . An apparatus for the crystal growth of biomacromolecules which crystallizes a biomacromolecule with a precipitant to yield the crystal of the biomacromolecule, comprising: 
 (i) a first compartment for accommodating a first fluid containing a biomacromolecule to be crystallized;    (ii) a second compartment for accommodating a second fluid containing a precipitant;    (iii) a concentration adjusting means for adjusting the concentration of the biomacromolecule in said first fluid and the concentration of the precipitant in said second fluid; and    (iv) a detection means for continuously detecting the crystal growth of said biomacromolecule in the first fluid in said first compartment.    
     
     
         2 . The apparatus for the crystal growth of biomacromolecules according to  claim 1 , wherein said concentration adjusting means has a dialysis means that is provided at the boundary between the first and second compartments and which establishes solution communication between the first and second fluids.  
     
     
         3 . The apparatus for the crystal growth of biomacromolecules according to  claim 1 , wherein said detection means is a visual detection means and has a light transmitting window provided on the first compartment such that light passes through the first compartment to enable visual detection of the crystal growth in it.  
     
     
         4 . The apparatus for the crystal growth of biomacromolecules according to  claim 3 , wherein said detection means has a polarizing filter on the optical path to said light transmitting window.  
     
     
         5 . The apparatus for the crystal growth of biomacromolecules according to  claim 4 , wherein said detection means has a light source and a detection unit for detecting the light issued from the light source, the polarizing filter comprises a first polarizing filter element that polarizes the light from the light source before it illuminates the light transmitting window and a second polarizing filter element that polarizes the light from the light source after it passes through the light transmitting window to travel to the outside of the first compartment, said detection means being so constructed that it detects the light from the light source after it has passed through the first polarizing filter element to be launched into the light transmitting window on the first compartment, through which it passes to emerge from the light transmitting window and then passes through the second polarizing filter element.  
     
     
         6 . The apparatus for the crystal growth of biomacromolecules according to  claim 5 , wherein said detection unit is an optical microscope, a CCD or a digital camera.  
     
     
         7 . The apparatus for the crystal growth of biomacromolecules according to any one of claims  1 - 6 , wherein the dialysis means is a semipermeable membrane that is not permeable to the biomacromolecule in the first fluid which is to be crystallized but is permeable to the precipitant in the second fluid.  
     
     
         8 . The apparatus for the crystal growth of biomacromolecules according to any one of claims  1 - 7 , which further includes a system temperature control means.  
     
     
         9 . A method of producing the crystal of a biomacromolecule using the apparatus according to  claim 1  for the crystal growth of biomacromolecules, said method comprising the steps of detecting the crystal growth of a biomacromolecule over time by the aforementioned detection means to determine the correlation between the concentration of a precipitant, the concentration of the biomacromolecule and the grown crystal of the biomacromolecule, representing the determined correlation in graphic form to construct a basic crystal phase diagram, and growing the crystal of the biomacromolecule under optimum crystallizing conditions on the basis of the constructed crystal phase diagram.  
     
     
         10 . A method in which the crystal of a biomacromolecule to be crystallized is grown under optimum conditions by continuously changing the concentration of the biomacromolecule and that of a precipitant, said method comprising the steps of: 
 (a) increasing the concentration of the biomacromolecule and/or the concentration of the precipitant so that the solution containing said biomacromolecule is shifted to the supersaturated region of a phase diagram for that biomacromolecule, thereby causing it to form crystal nuclei; and    (b) thereafter adjusting the concentration of the biomacromolecule and/or that of the precipitant such that the solution containing said biomacromolecule is shifted to the metastable region of the phase diagram for that biomacromolecule, thereby growing the crystal nuclei formed in step (a).    
     
     
         11 . The method according to  claim 10 , which further includes step (c) of choosing only one crystal nucleus between steps (a) and (b).  
     
     
         12 . The method according to  claim 10  or  11 , wherein the biomacromolecule to be crystallized is a protein.  
     
     
         13 . The method according to  claim 10  or  11 , wherein the biomacromolecule to be crystallized is a nucleic acid.