Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
2001-11-02
2003-07-15
Meller, Michael V. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S195000, C435S219000, C435S189000, C435S190000, C435S201000, C435S209000, C435S816000, C435S814000, C435S193000
Reexamination Certificate
active
06593118
ABSTRACT:
FIELD OF INVENTION
The present invention relates to crystallization, and particularly to a method for obtaining protein crystals having a desired morphology.
BACKGROUND OF THE INVENTION
Intensive research efforts have been directed to the precipitation and crystallization of enzyme as a means of purification and preparation of enzyme products. For example, in U.S. Pat. No. 4,654,667, a process is disclosed for the recovery of an enzyme from solution by concentrating to supersaturation the enzyme-containing solution at pH near the isoelectric point of the enzyme, inducing crystallization and recovering the crystallized final product. Inducing crystallization is achieved by allowing the enzymes to spontaneously crystallize upon concentration or by seeding, sound, stirring or scratching the inner surface of the container. Crystallization of alpha-amylase is exemplified.
In PCT Publication No. WO 89/08703, a process is described for the crystallization of subtilisin by adding a halide salt, such as sodium chloride or calcium chloride, to a concentrated subtilisin solution of at least about 40 grams per liter.
In EP 506,866, a method for the crystallization of enzymes is disclosed which is characterized by using as a starting material an aqueous solution containing liquid with a relatively high enzyme purity and a concentration of enzyme of about at least 5 grams per liter and adding as a crystallization agent an easily soluble salt of the non-halide type to a concentration which is considerably smaller than the amount necessary to precipitate the enzymes in an amorphous form. Crystallization of certain subtilisin enzymes at temperatures up to 30° C. is exemplified. Sodium sulfate is used to help purify the protease product but not for crystallization.
In spite of these advances in the field of enzyme crystallization, inexpensive and efficient crystallization of proteases suitable for large scale production has remained problematic in industry. The ability to rapidly produce crystals with a desirable morphology at an industrial scale would represent a large savings and be of great importance to the industry.
SUMMARY OF THE INVENTION
One aspect of the present invention provides a crystallization process for rapidly obtaining crystals having a desired morphology (e.g., square plates, hexagonal or rectangular crystals, etc.). Typically, the desired morphology will be one wherein the crystals exhibit increased strength over other possible crystal morphologies (e.g., square or rectangular plates or cubes, as opposed to elongated needles or rods). In one embodiment, a starting temperature is selected such that square-plate crystals are obtained. The starting temperature can be, for example, a temperature below room temperature (e.g., less than 20 degrees C.). A temperature shift or increase is then effected, preferably in a manner to minimize or avoid further nucleation, such that the crystals continue to grow on the square plates but with different kinetics, e.g., a higher rate of crystallization. The temperature shift can be, for example, to at least room temperature (e.g., between about 20 and 60 degrees C.). As a result, the process gives a crystalline product with the desirable morphology at a higher crystallization rate.
The method of the present invention is especially useful in quickly obtaining crystals of a protein, such as an enzyme, having a desired morphology. In one embodiment, the method is used to realize at least about 90% crystallization in less than 25 hours from a enzyme-containing solution, with the enzyme crystals having a predominantly square-plate morphology.
Other features, aspects and advantages of the present invention will become apparent from the following detailed description, in conjunction with the drawings and appended claims.
REFERENCES:
patent: 4659667 (1987-04-01), Brewer et al.
patent: 5602232 (1997-02-01), Reichert et al.
patent: 506866 (1993-10-01), None
patent: WO 89/08703 (1989-09-01), None
patent: WO 91/09943 (1991-07-01), None
patent: WO 97/15660 (1997-05-01), None
patent: WO 97/33983 (1997-09-01), None
Copy of International Search Report for PCT/US00/05564.
Genencor International Inc.
Genencor International Inc.
Meller Michael V.
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