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WO2003080900A1 - Systeme et procede de cristallisation - Google Patents

Systeme et procede de cristallisation Download PDF

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Publication number
WO2003080900A1
WO2003080900A1 PCT/GB2003/001230 GB0301230W WO03080900A1 WO 2003080900 A1 WO2003080900 A1 WO 2003080900A1 GB 0301230 W GB0301230 W GB 0301230W WO 03080900 A1 WO03080900 A1 WO 03080900A1
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WO
WIPO (PCT)
Prior art keywords
temperature
array
wells
channels
crystallisation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2003/001230
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English (en)
Inventor
Neil Isaacs
Jon Cooper
Gabriela Juarez-Martinez
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University of Glasgow
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University of Glasgow
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Filing date
Publication date
Application filed by University of Glasgow filed Critical University of Glasgow
Priority to EP03715094A priority Critical patent/EP1490536A1/fr
Priority to AU2003219289A priority patent/AU2003219289A1/en
Publication of WO2003080900A1 publication Critical patent/WO2003080900A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C30B35/00—Apparatus not otherwise provided for, specially adapted for the growth, production or after-treatment of single crystals or of a homogeneous polycrystalline material with defined structure
    • C30B35/002—Crucibles or containers
    • 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 a system and method for optimising crystal growth and screening crystallisation conditions, the system and method being particularly useful for protein crystal growth.
  • NMR nuclear magnetic resonance
  • X-ray diffraction X-ray diffraction
  • An object of the present invention is to provide a system and method for improved screening and optimisation of protein crystallisation.
  • a crystallisation system comprising an array of wells or channels defined in a substrate, the wells or channels being for receiving crystallisation solution, and means for providing a temperature differential across the array.
  • the system is a protein crystallisation system.
  • the substrate may be any suitable medium that can be used to define wells or channels.
  • the substrate may comprise silicon.
  • the substrate may comprise a thermally conducting polymer, such as that currently marketed under the trade mark "Coolpoly” .
  • the array may comprise a 2D grid of wells or channels.
  • the wells or channels may have dimensions in the millimeter range, for example 3mm by 3mm.
  • Means may be provided for sealing the solution within the wells or channels, thereby to avoid evaporation.
  • the means for providing a temperature differential may be operable to cool and/or heat the array relative to ambient temperature.
  • the means for providing a temperature differential may comprise one or more peltier elements .
  • a controller may be provided for controlling the temperature across the array.
  • the controller may include temperature sensors for measuring temperature across the array.
  • the temperature sensors may be thermistors or thermocouples .
  • the temperature sensors may be in direct thermal contact with the substrate.
  • the controller may be operable to maintain a pre-determined temperature change or gradient across the array. This may be done using feedback from the peltier elements and the temperature sensors using one or more temperature controllers.
  • the temperature may change linearly as a function of position across the array.
  • a crystallisation system comprising an array of wells or channels for receiving crystallisation solution and means for providing a temperature differential across at least part of the array, the arrangement being such that at least some of the wells or channels are aligned substantially perpendicular to a direction in which the temperature is to be changed.
  • the system is a protein crystallisation system.
  • the array may comprise a 2D grid of wells or channels.
  • the wells or channels may have dimensions in the millimeter range, for example 3mm by 3mm. For microchannels the length could be in cm range but the width and depth in the micrometer or mm range.
  • the array may be defined in a substrate.
  • the substrate may comprise silicon.
  • the substrate may comprise a thermally conducting polymer, such as that marketed under the trade mark "Coolpoly" .
  • a method for determining optimal crystal growth conditions comprising introducing into an array of wells or channels defined in a substrate a crystallisation solution, and maintaining a temperature differential across the array. In this way, many samples of the solution can be tested simultaneously at different temperatures.
  • the method may further comprise sealing or isolating the crystallisation solution within the wells or channels, thereby to limit evaporation.
  • Figure 1 is a system for crystallising proteins
  • Figure 2 is a flow diagram that shows schematically a method for fabricating an array of microchambers for use in the system of Figure 1;
  • Figure 3 is a photograph of a crystallisation array made using the method of Figure 2;
  • Figure 4 is a plot of temperature variation along the centre of the device of Figure 1 ;
  • Figure 5 is a plot of 2D temperature distribution measured across half of the device of Figure 1, in which the cross-hatched boxes indicate the relative positions of two peltier elements;
  • Figures 6 (A) to (D) are micrographs of protein crystals obtained with the crystallisation device using a first set of crystallisation conditions;
  • Figures 7 (A) to (D) are micrographs of protein crystals obtained with the crystallisation device using a second set of crystallisation conditions;
  • Figures 8 (A) to (D) are micrographs of protein crystals obtained with the crystallisation device using a third set of crystallisation conditions, and
  • Figure 9 is a diffraction pattern from a lysozyme crystal .
  • Figure 1 shows an arrangement 10 for crystallising proteins having a silicon wafer 12 that includes an array
  • array it is meant any ordered arrangement of wells, and could include a single row of such wells.
  • the array of wells 14 is arranged as a 2D grid.
  • a 5 by 5 grid is shown, although for the crystallisation experiments discussed later a 10 by 10 grid was used.
  • the dimensions of each chamber are 3mm wide by 3 mm long by 1.1mm deep .
  • the separation of adjacent chambers 16 is 2mm, so that the centre to centre spacing is 5mm. These dimensions could of course be varied as desired.
  • Mounted on opposing sides of the wafer 12 are two peltier elements 18 for creating a temperature gradient across the array 14.
  • the direction of heat flow caused by the peltier elements 18 across the array 14 is shown in Figure 1. From this it can be seen that the array 14 is located relative to the peltier elements 18 in such a manner that rows of the wells 16 are aligned substantially perpendicular to the direction of heat flow.
  • the wafer 12 and the peltier elements 18 are all carried on a heat sink 20.
  • two thermistors 22 are provided in thermal contact with the wafer 12, one on either side of the array 14.
  • a control module 24 Connected to each of the thermistors 22 and the peltier elements 18 is a control module 24. This is operable to set a desired temperature gradient by sending appropriate control signals to the peltier elements 18.
  • the control module 24 is also adapted to monitor the temperature on either side of the array 14 using information from the thermistors 22.
  • the control module 24 is operable to identify if the predetermined gradient has been achieved and whether there are any changes in temperature across the array 14 over time. In the event that the desired temperature differential has not been achieved or the temperature does drift with time, the control module 24 is operable to send appropriate control signals to the peltier elements 18 in order to modify their output. In this way, heat flow and so the temperature differential can be maintained substantially constant over time.
  • the array of Figure 1 can be fabricated using standard photolithography techniques, reactive ion etching (RIE) and anisotropic wet etching.
  • Figure 2 shows the process steps for fabricating the microchamber array used in Figure 1.
  • the wafer 12 to be processed is, for example, a 3 inch diameter ⁇ 100> single crystal silicon wafer 26, being 2 mm thick with 100 nm of low pressure chemical vapour deposited (LPCVD) silicon nitride 28 on both sides.
  • LPCVD low pressure chemical vapour deposited
  • a layer of positive photoresist 30 (S1818 from Microposit) is spun onto one side of the wafer at 1000 rpm.
  • the template for the microchamber array is defined in the photoresist 30 using UN exposure through a corresponding photomask.
  • the template defines a 10 by 10 grid of squares, each being 3mm by 3mm and being separated by 2mm.
  • the template or pattern is subsequently transferred into the Si 3 ⁇ 4 layer by development of the photoresist, thereby to provide a masking layer 32.
  • the masked substrate is then etched.
  • the etch was done for 8 minutes using a reactive ion etch (RIE) that can be done using an Oxford Plasma RIE-80 with a C 2 F 6 etch a flow meter reading of 80%, corrected flow 20 seem and an RF power of 100 W.
  • RIE reactive ion etch
  • the wafer is wet etched. This can be done using a 40% aqueous solution (v/v) potassium hydroxide (Micro Image Technology Ltd. 215-181-3) .
  • the final etch depth can be controlled, and in the present case was selected to be 1.1mm. Care should be taken during the process of silicon micromachining to control the temperature of the wet etch bath in order to ensure uniform and reproducible depths for each of the chambers.
  • the remaining Si 3 N 4 on both sides of the wafer is removed using the RIE procedure described above. The end result is a 10 by 10 grid of chambers 16, each chamber having dimensions of substantially 3mm by 3mm by 2mm and a total
  • the microchambers 16 of the array 14 of Figure 1 are each filled with protein in solution, the solution for each chamber being the same.
  • the chambers 16 are sealed to avoid evaporation.
  • the peltier elements 18 are then used to establish a varying temperature across the array. This is done by setting the elements 18 to different temperatures, thereby causing a net heat flow across the substrate. As will be appreciated, the magnitude of the heat flow and so the temperature gradient depends on the temperatures at which the peltier elements 18 are set. The temperature may vary linearly across the array, as shown in Figure 4.
  • the thermistor signals were logged using a data acquisition system. Analysis of this data confirmed a
  • C is a constant of integration subject to ambient conditions. Heat losses will occur at the perimeter of the device, which in a regulated environment will add a corrective constant term to the value of Q, in this case x. As expected, at the edges of the wafer the gradient is less uniform, and in practice, measurements are best made in the centre of the array. In a working device, such deviations could be readily compensated for either in the physical design, or experimentally.
  • the first solution tested was hen egg white lysozyme from Fluka (Rieden-de Haen) , sodium acetate (S-9513) and sodium nitrate (22,134-1), both purchased from Sigma Chemical Co. All other reagents, unless stated were from Aldrich. The protein/agent mix should be such that supersaturation is instantaneously reached. Where appropriate, the solutions were prepared with de-ionised water from a Millipore Elix 10 system and subsequently
  • the samples were prepared in Eppendorf tubes by adding equal volumes of a solution of lysozyme (at a final concentration of 30 mg ml "1 ) and the crystallisation agent, sodium nitrate, with a range of final concentrations varying in a step-wise manner between 33mM and 800mM. All solutions were equilibrated in an acetate buffer, pH 4.5. Once the final crystallisation solutions were made, each of the microchambers of the array was filled with
  • the peltier elements were set up to maintain the temperature at one end of the array at 12C and the other end at 40C.
  • the direction of heat flow across the device is shown in Figure 1.
  • the resultant temperature gradient was maintained constant for twelve days, so that over this period the temperature of individual chambers was held at a substantially constant value.
  • neighbouring chambers may be at different temperatures.
  • Figure 6 (A) to (D) Clear morphological differences can be observed in Figure 6 (A) to (D) as a function of the temperature gradient across the device at constant concentration of the crystallisation.
  • Figure 6 (A) shows growth at relatively low temperatures. At this level, rod-like clusters appear.
  • Figures 6 (B) to (D) show crystals that were grown at increasing temperatures. As can be seen from these, the rod-like clusters gradually transform to single crystals as the temperature gets higher. Hence, by running a single experiment the temperature dependence of the crystal growth can be determined, within a reasonable degree of accuracy.
  • Figures 7 (A) to (D) show micrographs of protein crystals, which were grown at 14.4+ 0.8°C using different concentrations of crystallisation agent (NaN0 3 ) . More specifically, Figure 7 (A) shows growth at a concentration of 0.30M; Figure 7
  • Figure 8 (A) to (D) show micrographs of the protein crystals grown with the same concentrations as for those of Figures 7 (A) to (D) , except in this case the crystals were grown at 18.2 ⁇ 0.6°C.
  • a crystal from the condition shown in Figure 7 was chosen for X-ray diffraction analysis.
  • Diffraction data was collected at room temperature for the crystal mounted in the capillary. This was done using an X-ray source, e.g. Nonius FR591 Rotating Anode Generator and an image plate detector MacScience DIP2000.
  • Figure 9 shows an example of the diffraction pattern from the selected lysozyme crystal.
  • the resolution of this is 1.78A at the edge of the detector. 180 frames were collected (1 deg, 20min exposure per image), which produced 20163 unique reflections. The average I/ ⁇ for all data was 7.8, the average redundancy 3.6, the overall completeness 99.7% and Rmerge 8.3%. Processing of the data was performed with the programs from the HKL suite of programs. This data demonstrates that the crystals produced are suitable for high quality X-ray diffraction analysis . Other experiments have been conducted to optimise the growth of non-model proteins, e.g. the C-terminal fragment of the tetanus toxin.
  • a protein was found to crystallise at between 18 and 19C but not at 21.5, at which temperature needle clusters appeared, these being unsatisfactory for structure determination.
  • Using the array of Figure 1 demonstrated quickly that this particular protein was very sensitive to changes in temperature, more so than had been previously realised. Prior attempts to crystallise the protein in a reproducible manner had failed. Identifying that the temperature sensitivity was greater than anticipated and finding the crystallisation temperature, enabled optimal growth conditions to be identified relatively quickly. Once the conditions were determined, it was possible to grow high quality protein crystals in a reproducible manner. This is advantageous.
  • the pattern was subsequently transferred into the silicon wafer by development of the photoresist, followed by inductive couple plasma, for 250min (ICP-Surface Technology System, Gases: SF6/C4F8/02, flow meter reading: 13s/7s/13s, corrected flow 130/85/10 seem, RF power: 600/12W) .
  • the wafer was cleaned with a solution of hydrogen peroxide and sulphuric acid in a ratio 1:7. This method etches the silicon quite fast.
  • the mask made of SU8-50 survives the process but needs to be removed after the ICP process with a solution of hydrogen peroxide and sulfuric acid 1:7.
  • other fabrication options exist .
  • the computer program could also be operable to load the substrate into a heat control unit, such as an incubator, and then use the incubator to control the temperature differential.
  • a heat control unit such as an incubator
  • the incubator may be adapted to receive a large number of substrates and may be able to create a range of temperature differentials across these.
  • the system described with reference to the drawings uses a silicon substrate for the protein crystallisation array.
  • silicon has some clear advantages, including the fact that there are established procedures for its micromachining, enabling a robust and reproducible fabrication process. Silicon has a thermal conductivity of -150W m "1 K "1 at 20°C, which makes it a good substrate for devices that require a rapid and efficient heat transfer.
  • a silicon microarray can be readily cleaned and re-used, although care must be taken in the cleaning protocol to avoid contamination between proteins (commercial solutions for removing proteins are readily available) .
  • microarray The use of silicon does however present some disadvantages, namely the high cost of non-standard wafers and the lack of optical transparency (which makes observation of the protein crystals difficult, without the addition of a dye) .
  • other material may be used for fabricating the microarray.
  • thermally conducting polymers for example the polymer sold under the trade mark "Coolpoly" .
  • Figure 1 shows wells or chambers that are defined in a substrate, microfluidic channels could also be used for receiving crystallisation solution.
  • the systems and methods in which the invention is embodied provide an improved mechanism for screening protein crystallisation.
  • the ability to screen the conditions of crystallisation and define the optimal parameter space increases the probability of finding new crystallisation conditions.
  • the systems and methods described herein are particularly valuable in optimising the conditions for intractable biological systems, including those such as membrane-bound proteins which are not only difficult to purify in large amounts, but also are difficult to crystallise.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Materials Engineering (AREA)
  • Metallurgy (AREA)
  • Organic Chemistry (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

L'invention concerne un système (10) de cristallogenèse, de préférence de cristaux bio-macromoléculaires (y compris de cristaux protéiques, sans exclusion d'acides nucléiques, etc.), qui présente un réseau (14) de puits ou canaux (16) délimité dans un substrat, lesdits puits ou canaux (16) étant destinés à recevoir une solution de cristallisation. Un régulateur de température (24) est associé au réseau (14) pour créer une différence de température aux bornes d'au moins une partie du réseau.
PCT/GB2003/001230 2002-03-22 2003-03-24 Systeme et procede de cristallisation Ceased WO2003080900A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP03715094A EP1490536A1 (fr) 2002-03-22 2003-03-24 Systeme et procede de cristallisation
AU2003219289A AU2003219289A1 (en) 2002-03-22 2003-03-24 A crystallisation system and method

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0206819.5 2002-03-22
GBGB0206819.5A GB0206819D0 (en) 2002-03-22 2002-03-22 A crystallisation system and method

Publications (1)

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WO2003080900A1 true WO2003080900A1 (fr) 2003-10-02

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PCT/GB2003/001230 Ceased WO2003080900A1 (fr) 2002-03-22 2003-03-24 Systeme et procede de cristallisation

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EP (1) EP1490536A1 (fr)
AU (1) AU2003219289A1 (fr)
GB (1) GB0206819D0 (fr)
WO (1) WO2003080900A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001051919A2 (fr) * 2000-01-07 2001-07-19 Transform Pharmaceuticals, Inc. Formation, identification et analyse a productivites elevees de formes solides diverses
US20020001538A1 (en) * 1998-08-13 2002-01-03 Damian A. Hajduk Multi-temperature modular reactor and method of using same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020001538A1 (en) * 1998-08-13 2002-01-03 Damian A. Hajduk Multi-temperature modular reactor and method of using same
WO2001051919A2 (fr) * 2000-01-07 2001-07-19 Transform Pharmaceuticals, Inc. Formation, identification et analyse a productivites elevees de formes solides diverses

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
J-KEM SCIENTIFIC, INC.: "Multi-Temperature Reaction Blocks", J-KEM ONLINE CATALOGUE, XP002250064, Retrieved from the Internet <URL:www.jkem.com/multitemp.html> [retrieved on 20030804] *
JUÁREZ-MARTÍNEZ G ET AL: "High-throughput screens for postgenomics: studies of protein crystallization using microsystems technology.", ANALYTICAL CHEMISTRY. UNITED STATES 15 JUL 2002, vol. 74, no. 14, 15 July 2002 (2002-07-15), pages 3505 - 3510, XP002250062, ISSN: 0003-2700 *
LUFT, JOSEPH R. ET AL: "Microbatch macromolecular crystallization on a thermal gradient", JOURNAL OF CRYSTAL GROWTH (1999), 196 (2-4), 447 -449, 1999, XP004154380 *
MAO HANBIN ET AL: "A microfluidic device with a linear temperature gradient for parallel and combinatorial measurements.", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 124, no. 16, 24 April 2002 (2002-04-24), April 24, 2002, pages 4432 - 4435, XP002250063, ISSN: 0002-7863 *

Also Published As

Publication number Publication date
GB0206819D0 (en) 2002-05-01
AU2003219289A1 (en) 2003-10-08
EP1490536A1 (fr) 2004-12-29

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