Crystals can be grown in forms suitable for x-ray diffraction studies.
A process for growing protein crystals encapsulated within membranes has been invented. This process begins with the encapsulation of a nearly saturated aqueous protein solution inside semipermeable membranes to form microcapsules. The encapsulation is effected by use of special formulations of a dissolved protein and a surfactant in an aqueous first liquid phase, which is placed into contact with a second, immiscible liquid phase that contains one or more polymers that are insoluble in the first phase. The second phase becomes formed into the semipermeable membranes that surround microglobules of the first phase, thereby forming the microcapsules. Once formed, the microcapsules are then dehydrated osmotically by exposure to a concentrated salt or polymer solution. The dehydration forms supersaturated solutions inside the microcapsules, thereby enabling nucleation and growth of protein crystals inside the microcapsules.
By suitable formulation of the polymer or salt solution and of other physical and chemical parameters, one can control the rate of transport of water out of the microcapsules through the membranes and thereby create physicochemical conditions that favor the growth, within each microcapsule, of one or a few large crystals suitable for analysis by x-ray diffraction. The membrane polymer can be formulated to consist of low-molecular-weight molecules that do not interfere with the x-ray diffraction analysis of the encapsulated crystals. During dehydration, an electrostatic field can be applied to exert additional control over the rate of dehydration.
This protein-crystal-encapsulation process is expected to constitute the basis of protein-growth experiments to be performed on the space shuttle and the International Space Station. As envisioned, the experiments would involve the exposure of immiscible liquids to each other in sequences of steps under microgravitational conditions. The experiments are expected to contribute to knowledge of the precise conditions under which protein crystals form. By enhancing the ability to grow crystals suitable for x-ray diffraction analysis, this knowledge can be expected to benefit not only the space program but also medicine and the pharmaceutical industry.
The prior art in osmotic dehydration for growing protein crystals involves the use of a small chamber in which a planar reverse-osmosis membrane is positioned between the mother liquor and a dehydrating salt solution. The prior art entails several disadvantages: (1) The nucleation and subsequent growth of protein crystals depend on increasing the concentration of precipitant and protein in the mother liquor; (2) there is no control over the effects of solute-driven convection on the surface of the crystal; (3) the crystals are not protected by any enclosure and thus are subject to physical damage as they are harvested and mounted; and (4) in some instances in the prior art, protein crystals have been protected by mounting them in aqueous gels, but this practice gives rise to the additional (as yet unsolved) problem of removing the gel material without adversely affecting the integrity of the protein crystals.
In contrast, the encapsulation of protein crystals in semipermeable membranes in the present process does not involve the use of gel, yet it creates closed environments that favor the growth of the crystals under prescribed conditions of controlled dehydration and protects the crystals against harsh environments that could otherwise damage the crystals.
In the present protein-crystal-encapsulation process, the microcapsules are spherical. The entire outer surface of the membrane of a microcapsule is accessible for osmotic dewatering as well as for and infiltration by hydrogen or hydroxyl ions. Such infiltration can be utilized to change the pH levels within microcapsules to favor or enhance protein saturation and subsequent crystal growth. The increase (relative to the prior art) in interfacial surface area occasioned by the transition from planar membranes to spherical microcapsules makes it possible to change conditions more rapidly throughout the mother liquor surrounding the crystal(s), thereby promoting the formation of more ordered and more nearly perfect crystals.
This work was done by Dennis R. Morrison of Johnson Space Center and Benjamin Mosier of the Institute for Research, Inc. For further information, access the Technical Support Package (TSP) free on-line at www.techbriefs.com/tsp under the Materials category.
This invention is owned by NASA, and a patent application has been filed. Inquiries concerning nonexclusive or exclusive license for its commercial development should be addressed to the Patent Counsel, Johnson Space Center, (281) 483-0837. Refer to MSC-22936.