Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-07-31
2003-07-08
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S259000, C435S410000
Reexamination Certificate
active
06589785
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to a method for disrupting biological material, in which the biological material is disrupted in the solid state and in the presence of a solid, denaturing substance.
When proteins, nucleic acids, fatty acids or other cell constituents are to be recovered, the cells have to be disrupted. A variety of methods and apparatus for disrupting cells have been developed, since the cells of various organisms differ in their behavior and, in some cases, can only be disrupted with difficulty. Also, the individual organisms or cells differ greatly with regard to the quality of the disruption. A particular problem when disrupting cells is the simultaneous liberation of degrading enzymes such as nucleases, proteases, lipases or glucosidases. In general, specific inhibitors are added to the batch to suppress such activities.
Usually, the cells are disrupted by means of ultrasound, French-press, high-pressure homogenizer or X-press while being suspended. To recover proteins, protease inhibitors such as, for example, PMSF, EDTA or leupeptin are generally added. However, the fact that the protease activity cannot always be suppressed sufficiently constitutes a disadvantage of these methods.
Solid cell material may also be comminuted in a mortar while cooling with liquid nitrogen, or in a vibration grinding mill (see, for example, Hess B. & Brand, K. (1983) Cell and Tissue Disintegration. General Aspects. In Methods Enzym. Anal., Third Ed., Eds. Bermeyer, H. U. VCI, Weinheim, FRG, Vol. 2, 26-30). However, the fact that the protein yields is relatively low compared with the other methods constitutes a disadvantage of this method.
To recover nucleic acids, the cells are generally disrupted by hydrolyzing the cell wall by means of lysozyme in the presence of SDS. The proteins are generally hydrolyzed by proteinase K. However, the fact that nucleic acids can only be recovered with difficulty from lysozyme-resistant cells constitutes a disadvantage of this method.
SUMMARY OF THE INVENTION
Object of the present invention was therefore to find a method which is widely applicable and allows cell constituents to be recovered in high yields.
Surprisingly, it has now been found that biological material can be disrupted readily in the solid state and in the presence of a solid, denaturing substance without degrading enzymes being activated.
Subject-matter of the present invention is therefore a method for disrupting biological material, in which the biological material is disrupted in the solid state and in the presence of solid, denaturing substance.
In a preferred embodiment, the solid, denaturing substance is a crystalline, preferably a crystalline organic, substance. Examples of especially preferred substances are urea, thiourea, guanidinium hydrochloride, guanidinium thiocyanate or ammonium sulfate. The disruption may also be carried out in the presence of various denaturing substances.
In general, the denaturing substance is employed in an approx. 1- to approx. 20-fold (w/w) excess, preferably in an approx. 1- to approx. 10-fold (w/w) excess and in particular in an approx. 1-fold (w/w) excess. It is furthermore advantageous for the biological material to be deep-frozen and preferably to remain deep-frozen during the disruption method according to the invention. Suitable for this purpose in an advantageous manner is, for example, liquid nitrogen.
In general, the biological material is disrupted by grinding, preferably in the presence, of a grinding ball.
Suitable as biological material is any biological material such as, for example, animal, human or plant cells, cell aggregates, tissues or animal, human or plant material. Also suitable are microorganisms such as fungi, bacteria, yeasts, protozoa or algae. Further suitable examples are
E. coli
, streptomycetes, Acremonium, Tetrahymena, Euglena, maize, wheat, muscle tissue or Actinoplanes.
To subsequently isolate the cell constituents such as proteins, nucleic acids (DNA, RNA), fatty acids, carbohydrates or the like after the disruption, the disrupted biological material is dissolved in a suitable buffer. The buffers which are customary for the cell type in question may be used for this purpose. The final concentration of urea is generally approx. 1 to approx. 10 M, preferably approx. 1 to approx. 5 M and especially approx. 4 M. It is advantageous to remove the cell debris and other solid constituents, for example by centrifugation, before the desired cell constituents are isolated by the methods known to the skilled worker.
DETAILED DESCRIPTION OF THE INVENTION
The method according to the invention is described hereinbelow in general terms and with reference to examples:
Usually, the organism or microorganism in question is grown in standard media known to the skilled worker. Thereafter, the cells can be harvested, for example by means of centrifugation. Then, they are usually washed and can be frozen for storage purposes. Plants can be grown on standard soils in the light or in the dark. Plant material is obtained, for example, by cutting of leaves, stalks or roots, which are usually immediately shock-frozen, for example in liquid nitrogen. Finally, the material can be comminuted mechanically by grinding in a nitrogen-cooled mortar.
The deep-frozen cell pellet, or the comminuted material, is then introduced into a shaking container consisting of, for example, teflon and cooled, for example using liquid nitrogen. The pellet is treated with, for example, urea in the abovementioned ratio. Advantageously, a grinding ball is then added, and the batch is shaken, for example in a laboratory vibration grinding mill, for example Dismembrator U, Braun Melsungen, Melsungen, FRG. Usually, the pellet is ground slowly in the presence of the urea by grinding at, for example, 2600 rpm, to give a powder. Advantageously, the batch is cooled with liquid nitrogen, and the procedure is repeated until a fine powder is obtained. After the disruption, the powder obtained is dissolved in a suitable buffer to the abovementioned final concentration of urea in order to isolate the cell constituents. The cell debris and other solid components are advantageously removed by centrifugation. The supernatant contains the desired cell constituents.
And important advantage of the method according to the invention is that cell constituents such as, for example, proteins, but also seaweed oil, can be recovered in high yields and in a simple manner, in particular from cells which are difficult to disrupt. In particular, it was possible to recover high-molecular weight (>20 kb) DNA was recovered in an advantageous fashion, for example from Actinoplanes, which are normally difficult to disrupt when using, for example, lysozyme. The DNA recovered in accordance with the invention is therefore also particularly suitable for isolating large DNA fragments. Also, optimization of the growing conditions for the microorganisms is generally not necessary in the method according to the invention.
The examples which follow are intended to illustrate the invention in greater detail without limiting it thereto.
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patent: 5128247 (1992-07-01), Koller
patent: 2002/0052026 (2002-05-01), Vicik
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Morinaga Milk Inc. Co., Ltd.,Patent Abstract of Japan 6:(122) : 57-050,884 Mar. 25, 1982.
Müllner Stefan
Neumann Thomas
Aventis Research & Technologies GmbH & Co KG
Connolly Bove & Lodge & Hutz LLP
Naff David M.
Ware Deborah K.
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