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WO2000000678A1 - Milieux de cristallisation - Google Patents

Milieux de cristallisation Download PDF

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Publication number
WO2000000678A1
WO2000000678A1 PCT/US1999/014390 US9914390W WO0000678A1 WO 2000000678 A1 WO2000000678 A1 WO 2000000678A1 US 9914390 W US9914390 W US 9914390W WO 0000678 A1 WO0000678 A1 WO 0000678A1
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WO
WIPO (PCT)
Prior art keywords
crystallization
solution
solutions
solution set
crystallization solution
Prior art date
Application number
PCT/US1999/014390
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English (en)
Inventor
Wim G. J. Hol
Steven H. Sarfaty
Lance J. Stewart
Hidong Kim
Original Assignee
University Of Washington
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Filing date
Publication date
Priority claimed from US09/150,629 external-priority patent/US6039804A/en
Application filed by University Of Washington filed Critical University Of Washington
Priority to AU47193/99A priority Critical patent/AU4719399A/en
Publication of WO2000000678A1 publication Critical patent/WO2000000678A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/06—Crystallising dishes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
    • C30B29/54—Organic compounds
    • C30B29/58—Macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions

Definitions

  • the present invention relates to solutions that are useful in the crystallization of molecules, especially macromolecules such as proteins. Background of the Invention
  • Macromolecular X-ray crystallography is an essential tool in modern drug discovery and molecular biology. Using X-ray crystallographic techniques, the three- dimensional structures of biological macromolecules, such as proteins, nucleic acids, and their various complexes, can be determined at practically atomic-level resolution from X-ray diffraction data.
  • One of the first and most important steps in the X-ray crystal structure determination of a target macromolecule is to grow large, well-diffracting crystals of the macromolecule.
  • crystal growth has become a rate- limiting step in the structure determination process.
  • Vapor diffusion is the most widely used technique for crystallization in modern macromolecular X-ray crystallography.
  • a small volume of the macromolecule sample is mixed with an approximately equal volume of a crystallization solution.
  • the resulting drop of liquid (containing macromolecule and dilute crystallization solution) is sealed in a chamber with a much larger reservoir volume of the crystallization solution.
  • the drop is kept separate from the reservoir of crystallization solvent either by hanging the drop from a glass cover slip or by sitting the drop on a pedestal above the level of the solvent in the reservoir.
  • the crystallization drop and the reservoir solutions equilibrate via vapor diffusion of volatile chemical species. Supersaturating concentrations of the macromolecule are achieved, resulting in crystallization of the macromolecule sample in the drop.
  • the present invention provides solutions (hereinafter referred to as crystallization solutions) useful for crystallizing proteins and other molecules, especially macromolecules.
  • the crystallization solutions of the invention are combined as four crystallization solution sets (identified herein as Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV).
  • Each of the four crystallization solution sets includes forty eight different crystallization solutions.
  • Each individual crystallization solution includes a precipitant and a buffer, and optionally includes at least one additive as set forth more fully herein.
  • compositions of the individual solutions that constitute Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV are set forth in Table I, Table II, Table III and Table IV, respectively.
  • the present invention is directed to sets of crystallization solutions including the solutions of one or more of Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV.
  • the present invention provides subsets of the crystallization solutions of Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV which are useful to determine crystallization conditions for a wide variety of molecules, especially biological macromolecules.
  • crystallization solution subsets are: subset I, solution numbers 1, 9, 10 and 28 of Crystallization Solution Set I; subset II, solution numbers 1, 8, 14, 26, 30 and 34 of Crystallization Solution Set II; subset III, solution numbers 2, 11, 18, 25, 26, 28, 29, 31, 38, 41 and 46 of Crystallization Solution Set III; and subset IV, solution numbers 2, 3, 5, 6, 7, 8, 17, 22, 24, 32, 34, 37, 43 and 48 of Crystallization Solution Set IV.
  • crystallization solution subsets preferably include at least one of subset I, subset II, subset III and subset IV.
  • kits including a plurality of crystallization solutions of the present invention and at least one crystallization plate that preferably includes a plurality of reservoirs.
  • the crystallization solutions of the crystallization solution set(s) are disposed within the reservoirs of the crystallization plates.
  • the presently preferred crystallization plates are disclosed in copending US Patent Application Serial Number 09/150,629, incorporated herein by reference.
  • kits including at least one crystallization plate and a set of crystallization solutions selected from the group of sets consisting of Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV.
  • kits including at least one crystallization plate and including a subset of crystallization solutions selected from the group of subsets consisting of: subset I, solution numbers 1, 9, 10 and 28 of Crystallization Solution Set I; subset II, solution numbers 1, 8, 14, 26, 30 and 34 of Crystallization Solution Set II; subset III, solution numbers 2, 11, 18, 25, 26, 28, 29, 31, 38, 41 and 46 of Crystallization Solution Set III; and subset IV, solution numbers 2, 3, 5, 6, 7, 8, 17, 22, 24, 32, 34, 37, 43 and 48 of Crystallization Solution Set IV.
  • the present invention provides solutions and kits that permit a large number of crystallization conditions to be easily and simultaneously tested in order to identify crystallization conditions under which a target molecule, especially a biological macromolecule such as a protein, can be crystallized.
  • a target molecule especially a biological macromolecule such as a protein
  • the crystallization solutions of the present invention have been successfully used to crystallize proteins regarded as recalcitrant to crystallization.
  • FIGURE 1 is a three dimensional view of a presently preferred crystallization plate useful for inclusion in a kit of the present invention.
  • FIGURE 2 is a view of the upper surface of a presently preferred crystallization plate useful for inclusion in a kit of the present invention. Detailed Description of the Preferred Embodiment
  • the present invention provides crystallization solutions useful for crystallizing proteins and other molecules, especially macromolecules.
  • the crystallization solutions of the invention are combined as four crystallization solution sets (identified herein as Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV).
  • Each of the four crystallization solution sets includes forty eight different crystallization solutions.
  • Each individual crystallization solution includes a precipitant, a buffer and optionally an additive.
  • the compositions of the individual solutions that constitute Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV are set forth in Tables I, II, III and IV, respectively.
  • CAPS 3-(cyclohexylamino)-l-propanesulfonic acid
  • CHES 2-(N- cyclohexylamino)ethanesulfonic acid
  • HEPES N-(2-hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid)
  • MES 2-(N-morpholino)ethanesulfonic acid
  • MME monomethyl ether
  • OAc - acetate
  • PEG polyethylene glycol
  • Tris tris(hydroxymethyl)aminomethane.
  • the present invention includes subsets of the crystallization solutions of Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV.
  • Presently preferred crystallization solution subsets are: subset I, solution numbers 1, 9, 10 and 28 of Crystallization Solution Set I; subset II, solution numbers 1, 8, 14, 26, 30 and 34 of Crystallization Solution Set II; subset III, solution numbers 2, 11, 18, 25, 26, 28, 29, 31, 38, 41 and 46 of Crystallization Solution Set III; and subset IV, solution numbers 2, 3, 5, 6, 7, 8, 17, 22, 24, 32, 34, 37, 43 and 48 of Crystallization Solution Set IV.
  • crystallization solution subsets preferably include at least one of subset I, subset II, subset III and subset IV.
  • crystallization solutions are made with ultrapure ASTM Type I water, and sterile-filtered into sterile tubes.
  • the sterile crystallization solutions should be stored at room temperature. The following stock solutions are utilized to formulate the crystallization solutions.
  • Acetate pH 4.5 1 M acetic acid and 1 M sodium acetate solutions are mixed together to make a pH 4.5 acetate stock solution. A 10-fold dilution of this stock solution is used in the final crystallization solution formulations, if required.
  • Cacodylate pH 6.5 A I M sodium cacodylate stock solution is adjusted to pH 6.5 with concentrated (37%) HC1. A 10-fold dilution of this stock solution is used in the final crystallization solution formulations, if required.
  • CHES pH 9.5 A 1 M CHES stock solution is adjusted to pH 9.5 with 50% NaOH. A 10-fold dilution of this stock solution is used in the final crystallization solution formulations, if required. Citrate pH 5.5. 0.5 M citric acid and 0.5 M sodium citrate solutions are mixed together to make a pH 5.5 citrate stock solution. A 5-fold dilution of this stock solution is used in the final crystallization solution formulations, if required.
  • HEPES pH 7.5 A 1 M HEPES stock solution is adjusted to pH 7.5 with 50% NaOH. A 10-fold dilution of this stock solution is used in the final crystallization solution formulations, if required.
  • Imidazole pH 8.0 A I M imidazole stock solution is adjusted to pH 8.0 with concentrated HC1. A 10-fold dilution of this stock solution is used in the final crystallization solution formulations, if required.
  • Na/K phosphate pH 6.2 0.5 M Na 2 HPO 4 and 0.5 M KH 2 PO 4 solutions are mixed together to make a pH 6.2 Na/K phosphate stock solution. A 5-fold dilution of this stock solution is used in the final crystallization solution formulations, if required.
  • Tris pH 7.0 (or pH 8.5).
  • a I M Tris base stock solution is adjusted to pH 7.0 or 8.5 with concentrated HC1. 10-fold dilutions of these stock solutions are used in the final crystallization solution formulations, if required.
  • the molecule When using the crystallization solutions of the present invention to crystallize a molecule, the molecule should be as highly purified as possible. If the molecule to be crystallized is a protein, preferably the protein should appear greater than 97% pure as determined by silver-stained SDS-PAGE.
  • the molecular sample is a biological macromolecule, such as a protein, the molecular sample should be in as minimal a buffer as possible (i.e., the buffer should contain as few chemical components as possible) to help maintain the biological activity of the macromolecule, and the molecular sample should preferably be at a concentration of 5-15 mg/ml.
  • the crystallization solutions of the present invention can be used in any crystallization technique.
  • Presently preferred crystallization techniques for use with the crystallization solutions of the present invention are vapor diffusion techniques as described, for example, in Gilliland, G.L. & Davies, D.R. (1984) Methods in Enzymol. 104:370-381; McPherson, A. (1990) Eur. J. Biochem. 189: 1-23 and Weber, P.C. (1991) Adv. in Prot. Chem. 41: 1-36.
  • hanging drop crystallization is a vapor diffusion technique that typically utilizes crystallization plates including a plurality of reservoirs, such as those available from Hampton Research (27632 El Lazo Rd., Georgia Niguel, CA 92677) and ICN-Flow (3300 Hyland Ave., Costa Mesa, CA 92626).
  • sealant such as petroleum jelly or vacuum grease, is applied to the rim of a crystallization plate reservoir and 0.5-1.0 ml of a single crystallization solution of the present invention is pipetted into the reservoir.
  • sitting drop crystallization is a type of vapor diffusion technique that utilizes sitting drop crystallization plates, including a plurality of reservoirs within each of which is located a pedestal that includes a sample depression within its upper end, such as those available from Charles Supper Co. (15 Tech Circle, Natick, MA 01760).
  • a sitting drop crystallization experiment utilizing the crystallization solutions of the present invention, 0.5-1.0 ml of a single crystallization solution are pipetted into a reservoir of a sitting drop crystallization plate and 1-10 ⁇ l of the sample are pipetted into the sample depression of the sitting drop pedestal. An equal volume of the crystallization solution that is in the reservoir is added to the sample drop and mixed.
  • the reservoirs can be individually sealed with sealant and cover slips, or the entire sitting drop plate can be sealed with a single piece of clear sealing tape after application of sample to all wells has been completed.
  • other crystallization techniques that can utilize the crystallization solutions of the present invention include sandwich drop vapor diffusion which is similar to hanging drop and sitting drop vapor diffusion, except that the crystallization drop is contacted on two sides by glass or plastic surfaces. See, e.g., A. McPherson, Eur. J. Biochem. 189: 1-23 (1990). Sandwich drop crystallization plates are available from Hampton Research and ICN-Flow.
  • the rate of equilibration by vapor diffusion can be modulated by placing a layer of oil between the crystallization drop and the reservoir (see, e.g., Chayen, N.E. (1997) J. Appl. Cryst. 30: 198-202).
  • oils can be used to seal microbatch crystallization drops, in the absence of a larger reservoir of crystallization solution (see, e.g., Chayen N.E. et al. (1990) J. Appl. Cryst. 23:297-302).
  • layers of sample solution and crystallization solution can be deposited in a capillary 0.5-1.0 mm in diameter, either with an air space between the solutions or with a direct liquid-liquid interface. Crystallization occurs by vapor diffusion or liquid-liquid diffusion inside the capillary.
  • the crystallization trials should preferably be stored in a place free of vibrations or mechanical shock, which could result in premature precipitation.
  • crystallization trials typically are recorded every one or two days.
  • the crystallization trials can be viewed under a stereo microscope at 10-lOOx magnification. If less than ten percent of the samples in the crystallization screen do not show heavy precipitate after one day, it may be desirable to increase the concentration of the sample molecule. If more than fifty percent of the samples in the crystallization screen show heavy precipitate after one day, it may be desirable to reduce the sample molecule concentration.
  • Crystals suitable for X-ray data collection are generally 0.1 mm or greater in their smallest dimension, and have clean, sharp edges. Viewing the crystallization trials between crossed polarizers often aids in distinguishing microcrystals from amorphous precipitate. Except for the rather unusual occurrence of a cubic space group, X-ray diffraction quality biological macromolecule crystals are birefringent (have more than one refractive index), and turn polarized light. When rotated between crossed polarizers, the intensity and/or color of light transmitted through birefringent crystals will change, with a periodicity of 90°. Amorphous precipitates will not transmit and turn polarized light.
  • the crystallization conditions may be optimized by adjusting parameters, such as pH and temperature.
  • Small crystals can be grown larger by seeding techniques (see, e.g., Thaller, C. et al. (1985) Methods in Enzymol. 114: 132-135; Stura, E.A. & Wilson, I.A. (1990) METHODS: A Companion to Methods in Enzymol. 1:38-49).
  • Using larger volume crystallization drops may also increase crystal size (see, e.g., Fox, K.M. & Karplus, P.A. (1993) J. Mol. Biol. 234:502-507).
  • Crystals should be harvested straight from the crystallization drop and mounted for data collection.
  • Crystallization Solution Set II have been specially formulated so that they will freeze as a clear amorphous glass, and not interfere with the diffraction of macromolecule crystals grown in these solutions.
  • crystals may be mounted directly from the crystallization drop, and frozen in liquid nitrogen or a stream of gaseous nitrogen for cryo-temperature X-ray diffraction data collection (as described, for example, in Rodgers, D.W. (1994) Structure, 2: 1135-1140).
  • crystallization conditions can be optimized, if necessary, by a variety of art-recognized techniques to generate very high quality crystals.
  • cryo-stabilizers such as glycerol, glucose, or PEG-400 can be added to the crystallization solutions of the present invention.
  • some macromolecule crystals may be damaged upon freezing, resulting in poor diffraction compared with their diffraction at room temperature.
  • cross-linking reagents such as glutaraldehyde may be used to make the crystals more resistant to mechanical stress upon freezing.
  • the four crystallization solution sets of the present invention have been shown to reproducibly crystallize proteins which are known to crystallize or are known to have a propensity to crystallize.
  • These model proteins include apoferritin, thaumatin, lysozyme and canavalin.
  • apoferritin is a large iron carrier protein in blood
  • thaumatin is an especially sweet protein from potato
  • lysozyme is from hen egg whites and is important for breaking down the glycoprotein shell of bacteria.
  • lysozyme crystallizes in solution number 28 of Crystallization Solution Set I; solution number 30 of Crystallization Solution Set II; solution numbers 28, 29, 31, 38 and 41 of Crystallization Solution Set III and in solution numbers 3, 7, 17, 24, 37, 43 and 48 of Crystallization Solution Set IV.
  • Canavalin crystallizes in solution number 10 of Crystallization Solution Set I; solution numbers 2, 26 and 46 of Crystallization Solution Set III and in solution numbers 5, 8, 22 and 34 of Crystallization Solution Set IV.
  • Thaumatin crystallizes in solution number 14 of Crystallization Solution Set II; solution numbers 18 and 38 of Crystallization Solution Set III and in solution number 37 of Crystallization Solution Set IV.
  • Apoferritin crystallizes in solution numbers 1 and 26 of Crystallization Solution Set II.
  • the crystallization solutions of the present invention have also been used to successfully crystallize human topoisomerase I, a protein which is notoriously difficult to crystallize.
  • the present invention provides kits including a plurality of crystallization solutions of the present invention and at least one crystallization plate that includes a plurality of reservoirs.
  • the crystallization solutions are disposed within the reservoirs of the crystallization plates which can therefore be immediately used to conduct crystallization experiments.
  • Any crystallization plates can be included in the kits of the present invention, including, by way of non-limiting example: Hampton Research plate models VDX, Linbro, Costar, Cryschem, Q-Plate, Q-Plate II and Crystal Clear Strips; Charles Supper Co. sitting drop plates and ICN Linbro model.
  • the presently preferred crystallization plates are disclosed in copending US Patent Application Serial Number 09/150,629, incorporated herein by reference.
  • crystallization tray 10 includes a body 12 having an upper surface 14, a lower surface 16, a first end 18, a second end 20, a first side 22 and a second side 24.
  • Body 12 defines a plurality of crystallization units 26.
  • Each crystallization unit 26 includes a central reservoir 28, four diffusion channels 30 and four drop chambers 32.
  • Each drop chamber 32 is connected to central reservoir 28 by one diffusion channel 30. While the plate of FIGURES 1 and 2 is preferred, other plates or arrays of vessels may suitably be employed with the solutions of the present invention.
  • kits including at least one crystallization plate and a set of crystallization solutions selected from the group of crystallization solution sets consisting of Crystallization Solution Set I, Crystallization Solution Set II, Crystallization Solution Set III and Crystallization Solution Set IV.
  • kits including at least one crystallization plate and a subset of crystallization solutions selected from the group of subsets consisting of: subset I, solution numbers 1, 9, 10 and 28 of Crystallization Solution Set I; subset II, solution numbers 1, 8, 14, 26, 30 and 34 of Crystallization Solution Set II; subset III, solution numbers 2, 11, 18, 25, 26, 28, 29, 31, 38, 41 and 46 of Crystallization Solution Set III; and subset IV, solution numbers 2, 3, 5, 6, 7, 8, 17, 22, 24, 32, 34, 37, 43 and 48 of Crystallization Solution Set IV.
  • a kit of the present invention may optionally include, for example, water- permeable silicone oil DC200 (BDH, Gallard Schlesinger Industries, 584 Mineola Ave., Carle Place, NY 11514-1744, Catalogue number #63002 4N), and/or paraffin oil (Fluka Chemical Corp., 980 South 2nd St., Ronkonkoma, NY 11779-7238, catalogue number #76235) which are useful in microbatch crystallizations, and vapor diffusion crystallizations with oils.
  • DC200 water- permeable silicone oil
  • paraffin oil Fluka Chemical Corp., 980 South 2nd St., Ronkonkoma, NY 11779-7238, catalogue number #76235
  • Protein slurry was dissolved by the addition of 1 M ammonium hydroxide until the solution appeared transparent.
  • the sample was then adjusted to 200 mM sodium chloride by the addition of 5 M sodium chloride stock solution.
  • the pH of the sample was then adjusted to pH 7.0 with the addition of 0.1 M hydrochloric acid.
  • the final concentration of protein was determined to be 30 milligrams per milliliter. This final protein solution was used in crystallization trials with Crystallization Solution Sets I, II, III and IV.
  • the apparatus used for the experiments was the Compact Crystallization Plate (Emerald Biostructures, 7869 N.E. Day Rd. W., Bainbridge Island, WA 98110).
  • Crystallization experiments were set up as follows. 0.2 milliliters of crystallization solution were placed into individual reservoir chambers of a Compact Crystallization Plate. Two microliters ( ⁇ l) of the crystallization solution were removed from the reservoir chambers and placed into individual drop chambers that are connected to the reservoir chamber by a vapor diffusion channel. 2 ⁇ l of the dissolved protein were then mixed with the 2 ⁇ l of crystallisation solution in the drop chamber. The crystallization chambers were sealed with Crystal Clear tape (Manco, Inc., 32150 Just Imagine Drive, Avon OH 44011), and the plates were maintained at 14°C. Crystals formed within twelve to sixteen hours. Canavalin crystallized in solution number 10 of Crystallization Solution Set I; solution numbers 2, 26 and 46 of Crystallization Solution Set III and in solution numbers 5, 8, 22 and 34 of Crystallization Solution Set IV.
  • Example 2 Crystallization of Lysozyme Purified lysozyme (obtained from hen egg whites) was purchased from Sigma-
  • Crystallization Solution Sets I, II, III and IV exactly as described in Example 1 for Canavalin. Crystals formed within four to five days. Lysozyme crystallized in solution number 28 of Crystallization Solution Set I; solution number 30 of Crystallization Solution Set II; solution numbers 28, 29, 31, 38 and 41 of Crystallization Solution Set III and in solution numbers 3, 7, 17, 24, 37, 43 and 48 of Crystallization Solution Set IV.
  • Apoferritin Horse Spleen
  • Catalogue Number A4890 The protein solution was at 25 mg/ml and was used directly in crystallization trials with Crystallization Solution Sets I, II, III and IV exactly as described in Example 1 for Canavalin. Crystals formed within two to five days. Apoferritin crystallized in solution numbers 1 and 26 of Crystallization Solution Set II.
  • Example 4 Crystallization of Thaumatin Purified Thaumatin (purified from Thaumatococcus danielli ⁇ ) was purchased from Sigma- Aldrich Co. (Caalogue number T7638). The protein powder was dissolved in water to a final concentration of 25 mg/ml. This final protein solution was used in crystallization trials with Crystallization Solution Sets I, II, III and IV exactly as described in Example 1 for Canavalin. Crystals formed within three to five days. Thaumatin crystallized in solution number 14 of Crystallization Solution Set II; solution numbers 18 and 38 of Crystallization Solution Set III and in solution number 37 of Crystallization Solution Set IV.
  • Example 5 Crystallization of Topoisomerase I Purified Human Topoisomerase I (70 kDa, amino acids 175 to 765 of the human topoisomerase I disclosed in P. D'Arpa et al., Proc. Nat'l Acad. Sci. U.S.A. 85: 2543-2547 (1988), with an active site mutation of amino acid 723 from tyrosine to phenylalanine) was used in a crystallization trial with Crystallization Solution Sets I, II, III and IV as follows.
  • the human Topoisomerase I was at 10 mg/ml in storage buffer (10 mM Tris-hydrochloric acid pH 7.5, 1 mM EDTA, 1 mM DTT).
  • 0.6 milliliters of crystallization solution were placed into individual reservoir chambers of a Cryschem sitting drop crystallization plate (Charles Supper & Co., 15 Tech Circle, Natick, MA 01760). 2 ⁇ l of the topoisomerase I solution were placed into individual sitting drop chambers of the Cryschem plate. 1 ⁇ l of a 22 base-pair duplex oligonucleotide (0.2 mM duplex oligonucleotide in 6 mM sodium chloride) was then mixed with the 2 ⁇ l of Topoisomerase I.
  • the sequence of the 5' to 3' strand of the duplex oligonucleotide is set forth in SEQ ID NO:l, and the sequence of the 3' to 5' strand of the duplex oligonucleotide is set forth in SEQ ID NO:2.
  • 3 ⁇ l of crystallization solution were then added to the topoisomerase-oligonucleotide mixture.
  • the crystallization chambers were sealed with Crystal Clear tape, and the plates were maintained at 20°C. Crystals formed within three to five days.
  • topoisomerase-oligonucleotide complex crystallized in solution number 25 of Crystallization Solution Set III, and in solution number 32 of Crystallization Solution Set IV. While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

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Abstract

Cette invention concerne, sous un premier aspect, des solutions de cristallisation qui sont utiles afin de cristalliser des protéines et d'autres molécules, notamment des macromolécules. Dans un mode de réalisation préféré, ces solutions de cristallisation sont regroupées en quatre ensembles de solutions de cristallisation. Chacun de ces quatre ensembles comprend quarante-huit solutions de cristallisation différentes. Chaque solution de cristallisation individuelle comprend un réactif précipitant, un tampon et, éventuellement, un ou plusieurs additifs. Sous un autre aspect, cette invention concerne des trousses comprenant plusieurs des solutions de cristallisation susmentionnées, et au moins un plateau de cristallisation qui comprend de préférence plusieurs réservoirs. Les solutions de cristallisation sont de préférence disposées dans les réservoirs des plateaux de cristallisation.
PCT/US1999/014390 1998-06-26 1999-06-25 Milieux de cristallisation WO2000000678A1 (fr)

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US9081298P 1998-06-26 1998-06-26
US9081198P 1998-06-26 1998-06-26
US60/090,811 1998-06-26
US60/090,812 1998-06-26
US09/150,629 1998-09-09
US09/150,629 US6039804A (en) 1998-09-09 1998-09-09 Crystallization tray

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Cited By (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003078700A1 (fr) * 2002-03-05 2003-09-25 Alfred Nordheim Dispositif d'application de substances liquides et procede correspondant
US6913732B2 (en) 2001-03-19 2005-07-05 Corning Incorporated Microplate for performing crystallography studies and methods for making and using such microplates
WO2006007702A1 (fr) * 2004-07-16 2006-01-26 Nextal Biotechnologie Inc. Procede et appareil pour l'optimisation de conditions de cristallisation d'un substrat
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