+

WO2003012430A1 - Utilisation de colorant pour distinguer un sel et des cristaux de proteines dans des conditions de microcristallisation - Google Patents

Utilisation de colorant pour distinguer un sel et des cristaux de proteines dans des conditions de microcristallisation Download PDF

Info

Publication number
WO2003012430A1
WO2003012430A1 PCT/US2002/024032 US0224032W WO03012430A1 WO 2003012430 A1 WO2003012430 A1 WO 2003012430A1 US 0224032 W US0224032 W US 0224032W WO 03012430 A1 WO03012430 A1 WO 03012430A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
crystals
component
dye
solution
Prior art date
Application number
PCT/US2002/024032
Other languages
English (en)
Inventor
Larry Cosenza
Terry Bray
Lawrence J. Delucas
Thomas Gester
David T. Hamrick
Original Assignee
Uab Research Foundation
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Uab Research Foundation filed Critical Uab Research Foundation
Publication of WO2003012430A1 publication Critical patent/WO2003012430A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N25/00—Investigating or analyzing materials by the use of thermal means
    • G01N25/14—Investigating or analyzing materials by the use of thermal means by using distillation, extraction, sublimation, condensation, freezing, or crystallisation
    • G01N25/147—Investigating or analyzing materials by the use of thermal means by using distillation, extraction, sublimation, condensation, freezing, or crystallisation by cristallisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
    • 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

  • This invention relates generally to the method of using detectable agents in crystallization of proteins, wherein the detectable agents are preferentially incorporated in the crystals of proteins or preferentially not incorporated in the crystals of proteins such that protein crystals can be readily distinguished from crystals of other substances which can form under the crystallization conditions, thereby allowing rapid and straightforward characterization of crystals and evaluation of crystallization conditions used to obtain the crystals.
  • this invention relates to a method of using dyes to facilitate the characterization of protein crystals present in volumes of one micro liter or less. Specifically, it relates to the crystallization of proteins from protein solutions containing dyes under conditions wherein the proteins which crystallize and the other components of the protein solutions which crystallize can be distinguished from one another on the basis of whether or not they form dyed crystals.
  • this invention relates to a method for forming a dyed protein crystal including the steps of: (1) providing a protein solution, wherein the protein solution contains at least one dye, which dye is capable of dyeing at least a portion of protein crystals formed from the protein solution; (2) subjecting the protein solution to environmental conditions effective to form protein crystals; and (3) detecting the presence of dyed protein crystals, whereby the presence of the dyed protein crystals indicates the presence of protein crystals.
  • the dye can be cchhoosseenn from the group consisting of methylene blue, methylene green, Izit 1 and crystal violet.
  • the protein solution is partitioned from the atmosphere.
  • the partitioning can be done so as to lower the rate at which transfer of solvent from the protein solution occurs.
  • the protein solution can be partitioned from the atmosphere by overlaying the protein solution with an oil.
  • the overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL's oil 1 (a 1:1 mixture of paraffin and silicone oil).
  • AL's oil 1 a 1:1 mixture of paraffin and silicone oil.
  • the particular mixture of oil selected can be optimized for the rate at which transfer of solvent from a protein solution occurs when the protein solution is overlayed with a particular mixture of oil.
  • the amount of oil overlaying the protein solution can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL's oil 1 (a 1:1 mixture of paraffin and silicone oil).
  • Reagent is commercially available from Hampton Research, Inc. (www.hamptonresearch.com) be selected so as to optimize the rate of solvent transfer from the protein solution, in particular, from the protein solution to the oil and from the oil to the atmosphere.
  • the protein solution further includes a component selected from the group consisting of salts, buffers, precipitants, crystallization aids and any combination thereof.
  • the component selected can crystallize under the controlled environmental conditions to which the protein solution is subjected, thereby producing component crystals.
  • the conditions under which the component crystallizes can include the step or process of adding a precipitate solution.
  • the component crystals formed can be undyed component crystals.
  • any crystals formed in the provided protein solution can be detected. If crystals are detected, dyed protein crystals can be distinguished from undyed component crystals. The protein crystals and component crystals can be detected by microscopy.
  • this invention relates to a method for screening protein crystal growth conditions, including the steps of: (1) providing a set of at least two protein solutions, wherein the protein solutions contain a dye, which is capable of dyeing at least a portion of protein crystals formed from the protein solution and which does not dye a significant portion of component crystals formed from the protein solution; (2) subjecting the protein solutions to predetermined conditions, wherein the environmental conditions to which each member of the set of protein solutions is subjected is not identical to the environmental conditions to which another member of the set is subjected; and (3) detecting the presence or non-presence of dyed protein crystals and/or undyed component crystals, wherein the presence of the dyed protein crystals indicates the formation of protein crystals; and the presence of undyed component crystals indicates formation of component crystals.
  • the set of protein solutions consists of greater than 10, 50, 100, 250, 500, 1000, 1500, 2000 or 5000 protein solutions.
  • the dye can be chosen from the group consisting of methylene blue, methylene green, Izit 1 and crystal violet.
  • the protein solutions are partitioned from the atmosphere.
  • the partitioning can be done so as to lower the-rate at which transfer of solvent from the protein solutions occurs.
  • the protein solutions can be partitioned from the atmosphere by overlaying the protein solutions with an oil.
  • the overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL's oil 1 (a 1 :1 mixture of paraffin and silicone oil).
  • the particular mixture of oil selected can be optimized for the rate at which transfer of solvent from a protein solution occurs when the protein solution is overlayed with a particular mixture of oil.
  • the amount of oil overlaying the protein solution can be selected so as to optimize the rate of solvent transfer from the protein solution, in particular, from the protein solution to the oil and from the oil to the atmosphere.
  • the protein solutions further include a component selected from the group consisting of salts, buffers, precipitants, crystallization aids and any combination thereof.
  • the component selected can crystallize under the controlled environmental conditions to which the protein solution is subjected, thereby producing component crystals.
  • the conditions under which the component crystallizes can include the step or process of adding a precipitate solution.
  • the component crystals formed can be undyed component crystals.
  • any crystals formed in the provided protein solutions can be detected. If crystals are detected, dyed protein crystals can be distinguished from undyed component crystals. The protein crystals and component crystals can be detected by microscopy.
  • the detection of dyed protein crystals indicates a combination of environmental conditions to promote crystal growth of a protein in the provide protein solutions.
  • the dyed protein crystals can be evaluated in respect to protein crystal quality.
  • Evaluation of protein crystal quality can include consideration of criteria selected from the group consisting of, but not limited to, size of crystals, volume of crystals, intensity of coloration of crystals by dye, color of coloration of crystals by dye, sharpness of crystal edges, and crystal shape.
  • the suitability of crystallization conditions can include evaluation of the protein crystal quality of the crystals obtained by a certain set of environmental conditions.
  • this invention relates to a method of forming a dyed component crystal including the steps of: (1) providing a component solution, wherein the component solution contains at least one dye, which dye is capable of dyeing at least a portion of component crystals formed from the component solution; (2) subjecting the component solution to environmental conditions effective to form component crystals; and (3) detecting the presence of dyed component crystals, whereby the presence of the dyed component crystals indicates the presence of component crystals.
  • the component solution is partitioned from the atmosphere. When the component solution is partitioned from the atmosphere, the partitioning can be done so as to lower the rate at which transfer of solvent to or from the component solution occurs.
  • the component solution can be partitioned from the atmosphere by overlaying the component solution with an oil.
  • the overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL's oil 1 (a 1 :1 mixture of paraffin and silicone oil).
  • the particular mixture of oil selected can be optimized for the rate at which transfer of solvent to or from the component solution occurs when the component solution is overlayed with a particular mixture of oil.
  • the amount of oil overlaying the component solution can be selected so as to optimize the rate of solvent transfer to or from the component solution, in particular, from the component solution to the oil, or vice versa, and from the oil to the atmosphere, or vice versa.
  • the component solution further includes a protein.
  • the protein selected can crystallize under the controlled environmental conditions to which the component solution is subjected, thereby producing protein crystals.
  • the conditions under which the protein crystallizes can include the step or process of adding a precipitate solution.
  • the protein crystals formed can be dyed protein crystals.
  • any crystals formed in the provided component solution can be detected. If crystals are detected, protein crystals can be distinguished from component crystals on the basis that the protein crystals are undyed protein crystals and the component crystals are dyed component crystals. The protein crystals and component crystals can be detected by microscopy.
  • this invention relates to a method for screening protein crystal growth conditions including the steps of: (1) providing a set of at least two protein solutions, wherein the protein solutions contain a dye, which is capable of dyeing at least a portion of component crystals formed from the protein solution and which does not dye a significant portion of protein crystals formed from the protein solution; (2) subjecting the protein solutions to predetermined conditions, wherein the environmental conditions to which each member of the set of protein solutions is subjected is not identical to the environmental conditions to which another member of the set is subjected; and (3) detecting the presence or non-presence of undyed protein crystals and/or dyed component crystals.
  • the presence of the undyed protein crystals can indicate the formation of protein crystals and the presence of the dyed component crystals can indicate formation of component crystals.
  • the set of protein solutions consists of greater than 10, 50, 100, 250, 500, 1000, 1500, 2000 or 5000 protein solutions.
  • the protein solutions are partitioned from the atmosphere.
  • the partitioning can be done so as to lower the rate at which transfer of solvent from the protein solutions occurs.
  • the protein solutions can be partitioned from the atmosphere by overlaying the protein solutions with an oil.
  • the overlaying oil can be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL's oil 1 (a 1:1 mixture of paraffin and silicone oil).
  • AL's oil 1 a 1:1 mixture of paraffin and silicone oil.
  • the particular mixture of oil selected can be optimized for the rate at which transfer of solvent from a protein solution occurs when the protein solution is overlayed with a particular mixture of oil.
  • the amount of oil overlaying the protein solution can be selected so as to optimize the rate of solvent transfer from the protein solution, in particular, from the protein solution to the oil and from the oil to the atmosphere.
  • the overlaying oil used, or the amount of overlaying oil used, to partition any single protein solution from the atmosphere can be the same or can be different than the overlaying oil used to partition any other protein solution from the atmosphere.
  • the protein solutions can further include a component selected from the group consisting of salts, buffers, precipitants, crystallization aids and any combination thereof.
  • the component selected can crystallize under the controlled environmental conditions to which the protein solution is subjected, thereby producing component crystals.
  • the conditions under which the component crystallizes can include the step or process of adding a precipitate solution.
  • the component crystals formed can be dyed component crystals.
  • crystals formed in the provided protein solutions can be detected. If crystals are detected, protein crystals can be distinguished from component crystals on the basis that the protein crystals are undyed protein crystals and the component crystals are dyed component crystals. The protein crystals and component crystals can be detected by microscopy.
  • the detection of undyed protein crystals indicates a combination of environmental conditions to promote crystal growth of a protein in the provided protein solutions.
  • the undyed protein crystals can be evaluated in respect to protein crystal quality.
  • Evaluation of protein crystal quality can include consideration of criteria selected from the group consisting of, but not limited to, size of crystals, volume of crystals, intensity of coloration of crystals by dye, color of coloration of crystals by dye, sharpness of crystal edges, and crystal shape.
  • the method of screening the suitability of crystallization conditions can include an evaluation of the characteristics, numbers or types of component crystals formed.
  • one advantage provided by one aspect of the invention is that the use of the dye to distinguish between salt and protein crystals allows rapid screening of crystals formed in volumes of less than or equal to approximately one microliter to distinguish between those formed of salt and those formed of protein.
  • the methods known to the art at this time that are used to distinguish between crystals of salt and crystals of protein are not able to achieve this result.
  • Crystals grown from volumes of solution less than or equal to approximately one microliter are too small for these classical methods of analysis.
  • the salt and protein crystals formed are too small to crash or to manipulate effectively for x-ray bombardment.
  • addition of any feasibly manipulated volume of dye solution has significant effects on the composition of the solution containing the crystals and, therefore, significant effects on the crystals therein.
  • use of such small volumes requires that the crystals be grown in solutions partitioned from the atmosphere, generally by an overlaying oil, it is extremely difficult to add a dye reagent to protein solutions containing crystals.
  • Figure 1 depicts micrograph images of crystallization trials.
  • Fig. 1A Izit under oil.
  • Fig. IB NaCl under oil.
  • Fig. lC NaCl + dye under oil.
  • Fig. ID Lysozyme under oil.
  • Fig. IE Lysozyme + dye under oil.
  • Fig. IF Thaumatin imder oil.
  • Fig. 1G Thaumatin + dye under oil.
  • Fig. 1H Catalase under oil.
  • Fig. II Catalase + dye under oil.
  • Fig. 1 J Trypsin under oil.
  • Fig. IK Trypsin + dye under oil using CS15.
  • Fig. 1L Trypsin + dye under oil using CS16. hi each trial, the oil was AL's oil, 50:50 mixture of paraffin oil and silicone oil and the dye was Izit.
  • Figure 2 depicts a lysozyme crystal. This dyed protein crystal, at the lower left quadrant of the well, was grown in the presence of Izit and has incorporated blue dye.
  • Figure 3 depicts a pepsin crystal. This dyed protein crystal was grown in the presence of Izit and has incorporated blue dye.
  • Figure 4 depicts a ⁇ -lactoglobulin crystal. This dyed protein crystal was grown in the presence of Izit and has incorporated blue dye.
  • Figure 5 depicts micrograph images of additional crystallization trials.
  • Figs. 5 A-5D depict trials conducted using Izit w/o any added protein under various screening conditions.
  • 5E-5F depict trials with added ⁇ -lactoglobulin.
  • Figures 5G-5I depict trials with added myristol transferase.
  • Figure 5 J depicts trials with added pepsin.
  • Figures 5K and 5L depict trials with protein Renac2.
  • Figures 5M and 5N depict trials with protein ROB 1.
  • Figure 5 O depicts a trial with protein BIO.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • Optional or “optionally” means that the subsequently described event oi ⁇ circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • solution optionally containing precipitate means that the solution may or may not contain precipitate and that the description includes both a solution without precipitate and a solution with a precipitate.
  • Detectable means that the presence or absence of the molecule or species so described can be determined.
  • detecttable means that the presence or absence of a certain property or characteristic of a molecule or species so described can be determined.
  • a visually detectable characteristic or property of a species or object, such as a crystal includes, but is not limited to, the intensity, shade and hue of the species or object.
  • Dye means an agent which renders another molecule or species detectable by its action on or association with the other molecule or species.
  • a dye will refer to a visually detectable small molecule, which interacts with, i.e. stains or colors, another molecule.
  • dye as used herein, may also mean an agent that acts on another molecule to render it detectable.
  • Dye as used herein, may also mean an agent, which absorbs or emits electromagnetic radiation outside of the visible spectrum, e.g., electromagnetic radiation within the ultraviolet spectrum.
  • Environment or “environmental conditions,” as used herein, means the sum total of all the conditions and elements that make up the surroundings and influence the development of any species subject to the environment.
  • the environmental conditions of a protein in solution can be defined in part by description of a limited number of conditions and elements, including but not limited to the identity and abundance of atoms or molecules in solution, including but not limited to solvents, solutes and precipitates; other conditions such as but not limited to pH, temperature and pressure; and the magnitude and orientation of other forces, including but not limited to gravitational forces, magnetic forces and electrostatic forces.
  • the dye(s) used in the practice of the invention may have no effect on the crystallization of a protein contained in the solution. While not being bound by theory, it is contemplated that the nature of the interaction between the selected dyes and protein crystals is such that it has no effect on the structure, stability or growth characteristics of the protein crystals of interest.
  • the dye(s) used in the practice of the invention may have a negligible effect on the crystallization of proteins contained in the solution.
  • protein crystals formed in the presence of the dye may be functionally equivalent in respect to their use to determine the structure of the crystallized protein, even though the protein crystals grown in the presence of the dye can be distinguished from the protein crystals grown in the absence of the dye on the basis of criteria that can be used to evaluate protein crystal quality, including the size of crystals, volume of crystals, sharpness of crystal edges and crystal shape.
  • the dye(s) used in the practice of the invention may have a significant effect on the crystallization of proteins contained in the solution.
  • the protein crystals' growth may be significantly slower when the protein solution contains the dye or the protein crystals' growth may be significantly faster when the protein solution contains the dye.
  • Significant differences in the growth rate of protein crystals are those greater than 5, 10, 15, 25, 50, 75, 100, 500 or 1000 fold different.
  • the quality of the crystals may be significantly different, as measured by the criteria for determining a protein crystal's quality, for example, size of crystals, volume of crystals, intensity of coloration of crystals by dye, color of coloration of crystals by dye, shaipness of crystal edges, and crystal shape.
  • colored dye crystals can be formed in the absence of protein. It will also be recognized that under some conditions, for example, when using Izit dye under high salt conditions and at low pH, colored dye crystals can be formed in the absence of protein. It will also be recognized that under some conditions, certain buffer components or salts can crystal with the dyes used to form colored crystals. Formation of such colored dye-only or colored component crystals can be detected by screening for the formation of colored crystals in the absence of any added protein or biomolecule of interest. Detection of the presence or absence of such colored crystals under such conditions can be used by one of skill in the art to evaluate the authenticity of colored crystals formed under such conditions when protein or the biomolecule of interest is present.
  • Screening protein crystal growth conditions refers broadly to the process of providing at least two protein solutions and subjecting them to different conditions and then determining whether or not the conditions allow for the crystallization of proteins included in the protein solution.
  • the protein solutions unless otherwise indicated, contain the same protein species, which is the protein of interest.
  • the environmental conditions that a given protein solution is subjected to can differ.
  • the protein solutions can also contain different protein species.
  • the protein species contained in the different protein solutions differ, the protein species that differ may be either the protein of interest and/or other proteins.
  • the protein of interest as will be recognized by those of skill in the art, is the protein for which the evaluation of protein crystallization conditions is intended. In other words, if an investigator is seeking to establish suitable conditions for the crystallization of protein X, then protein X is the protein of interest.
  • the volume of a protein solution used in the method of forming dyed or undyed protein crystals or in screening protein crystallization conditions can be greater than 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800 or 900 pico-, nano-, or micro-liters.
  • the volume of a protein solution used in the method of forming dyed or undyed protein crystals or in screening protein crystallization conditions, as is described herein, can be less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 milli-, micro-, nano- or pico- liters.
  • Partitioned refers to the segregation of the protein solution from the atmosphere such that the transfer of at least one species present in the solution from the solution to the atmosphere occurs at a lower rate than when the protein solution is not partitioned from the atmosphere.
  • segregation of the protein solution from the atmosphere by the interposition of a semipermeable membrane, which allows the passage of certain solvent molecules, but not others would partition the protein solution from the atmosphere.
  • the partitioning may be done so as to lower the rate at which transfer of solvent from the protein solution occurs.
  • the protein solution may be partitioned from the atmosphere by overlaying the protein solution with an oil.
  • the overlaying oil may be selected from the group consisting of paraffin oil, silicone oil or a combination thereof, for example, AL's oil (a 1 : 1 mixture of paraffin and silicone oil).
  • the particular overlaying oil selected may be optimized for the rate at which transfer of solvent from a protein solution occurs when the protein solution is overlayed with a particular mixture of oil.
  • Paraffin oil is more restrictive to evaporation of water from the overlayed protein solution than either silicone oil or mixtures containing paraffin oil and silicone oil. Mixtures of the two oils can be used where the rate of evaporation to be allowed falls between the rate allowed when 100% paraffin oil is used and when 100% silicone oil is used.
  • the %(v/v) of either of the two oils, either the paraffin oil or the silicone oil can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%.
  • the application and use of the invention greatly decreases both the quantity of labor and protein required to screen potential crystallization conditions to obtain effective crystallization conditions to provide the suitable high quality crystals required for x-ray crystallography of biomolecules for the use in stracture based drag design and the attendant benefits thereof.
  • the method of the present invention produces a significant aid in determining the conditions suitable for crystallizing proteins, thereby greatly facilitating the production of suitable protein crystals for structural deteimination without the excessive expenditure of limited resources.
  • the presently disclosed method can facilitate a great reduction in the quantity of protein needed for the screening of large numbers of crystallization conditions and the time required to analyze the outcome of each crystallization condition trial. It does so by allowing rapid discrimination between crystals of protein and crystals of other components present in a crystallization trial in very small volumes. Furthermore, the ease in discrimination between protein crystals of interest and crystals of other materials (e.g., salt, buffer, precipitants) is of particular usefulness when the presently disclosed use of dye to distinguish between protein and salt crystals is applied to pico, nano or meso scale crystallization trials performed in a microarray or other micro-device.
  • materials e.g., salt, buffer, precipitants
  • the crystallization trials, or necessary parts thereof can be automated.
  • the method used to characterize the nature of crystals present does not require physical contact between the crystal of interest and a probe or other such element, as is required for testing of crystals using conventional means, the method is more amenable to automation than conventional methods now used to determine that a crystal is a protein crystal.
  • Embodiments of the method that are automated are contemplated and provide for the preparation and/or rapid analysis of many samples in high throughput applications.
  • Automated systems to form solutions for preparing crystallization trials, to conduct trials and to monitor the resulting crystallizations can include any of those features and aspects as described in U.S. provisional application 60/128,018 and U.S. utility application 09/543,326 both of which are incorporated herein by reference in their entirety.
  • the automated systems or components thereof need not include all features described in the previous applications.
  • a fully automated system need only include an automated dispensing system to deliver crystallization trial volumes to receptacles, a capability to provide an environment in which crystals can form under at least some conditions, and an ability to detect the presence or absence of formed crystals.
  • the present invention includes the use of dye to facilitate the detection of crystals.
  • devices of the present invention adapted for automated screening do provide solutions containing the appropriate selected dye and do provide an environment that allows detection of dyed crystals.
  • provision of that environment is effected by use of wells having clear sides or a clear path for optical detection.
  • such an environment need not provide such a clear optical path provided other means for detecting the presence of the dyed crystals is provided.
  • other means include, but are not limited to, detectors present within the wells, devices for removal of crystals from wells, and detectors as are known in the art that do not rely upon the presence of a clear optical path, but instead rely on other physical or chemical properties.
  • the method can include use of an automated system that dispenses the appropriate solutions to form a crystallization trial that includes the presence of a dye useful in the practice of the invention and overlays the crystallization trial with an oil.
  • an automated solution can include a microairay for crystallization trials, an automated dispensing mechanism for dispensing the solutions, an automated dispensing mechanism for dispensing the overlaying oil, and an automated means for detecting crystal growth.
  • the method can encompass use of an analysis station that detects the color of crystals in samples that are provided to the analysis station in an automated fashion.
  • detection of crystal color can include the monitoring and detection of selected portions of the spectra of electromagnetic radiation that are absorbed, reflected or transmitted by a crystal, whereby detection of said selected portions of the spectra are indicative of a color.
  • Such automated methods can further include the sorting of crystals in regard to their determined characteristics.
  • sorting can be of a physical nature (i.e., the samples containing the crystals are segregated according to the nature of crystals contained therein) or can be of an informational nature (i.e., the identity of samples containing crystals of a particular nature and/or the location of crystals of a particular nature within a sample are recorded).
  • Such automated methods can also include determination of the number of crystals or objects of specified character or identity within a given sample, set of samples or other groups. Further, the number and identity relating to obtained crystals can also be used as a descriptor of conditions used to obtain crystals. For example, the total number of biomolecule crystals obtained and/or the fraction of crystals obtained that are biomolecule crystals can be used to describe results obtained using specific sets of conditions that can be used to form crystals of a specified character.
  • the automated method can monitor samples or crystals within samples.
  • the automated method can operate in response to a predetermined program.
  • the predetermined program can include input or instructions from the user. Input or instructions can be provided prior to the screening process or can be provided during the screening process either in response to queries generated by the predetermined program or by the initiative of the user.
  • Data obtained from the method can include images and data sets representing images or data derived from both images or selected portions of images. Images can be acquired automatically, with user action or with a combination of both automated and non-automated processes. Particular details regarding details of data analysis and determination of whether a biomolecule or a nonbiomolecule (i.e., a protein crystal or a salt crystal) will, of course, vary depending on the characteristics of the crystallization conditions and the dye being used. Optimization of such particular details are well understood by those of skill in the ail and would be recognized not to rise to the level of undue experimentation.
  • a biomolecule or a nonbiomolecule i.e., a protein crystal or a salt crystal
  • this example includes the addition of a blue dye to crystallization trials of approximately one microliter or lower volumes under paraffin oil as an aid to the detection and the characterization of protein crystals and/or the characterization of crystals as protein crystals.
  • the proteins screened in this example were: hen egg white lysozyme (Boehringer Mannheim, 1243004), 30 mg/ml in 0.1 M sodium acetate (pH 4.7); thaumatin (Sigma, T7638), 30 mg /ml in 100 mM Tris-HCl (pH 6.5); bovine catalase (Sigma, C40 ), 30 mg / ml in 50 mM HEPES (pH 7.0); cellulase from T.
  • viride (Sigma, C40), 40 mg / ml in 100 mM Tris-HCl (pH 8.5); bovine trypsin (Sigma, T8253), 60 mg / ml in 25 mM HEPES (pH 7.0), 10 mM CaCl 2 , 10 mg / ml benzamidin HC1; porcine pepsin (Sigma, P6887), 60 mg / ml in 100 mM cacodylate buffer (pH 6.5), 0.2 M Ca acetate; equine seram albumin (Sigma, A3434), 50 mg / ml in 50 mM sodium acetate (pH 5.5); Bacillus lichenformis ⁇ -amylase (Sigma, A4551), 25 mg / ml in 50 mM cacodylate buffer (pH 6.75), 2 mM CaCl 2 ; Barley ⁇ -amylase (Sigma, A7130), 8 mg / ml in
  • Precipitants were prepared using reagents from Sigma or Hampton Research. Paraffin oil and Izit were purchased from Hampton Research. Izit is a commercial preparation of methylene blue dye, a planar aromatic dye with a molecular weight of 319 daltons. Crystallization trials were performed in Labsystems 384 clinical plates under 40 ⁇ l of paraffin oil.
  • Crystallization trials were initiated by mixing equal portions of protein and precipitant solutions prior to depositing a 0.5 ⁇ l protein solution drop in the bottom of a crystallization chamber. Protein solutions were then covered with 40 ⁇ l of paraffin oil. The crystallization chambers were left unsealed to incubate at 20 °C. Crystal growth was monitored daily.
  • test proteins crystallized in the absence of dye, specifically, lysozyme, thaumatin, cellulase, pepsin, trypsin, equine serum albumin and catalyse crystals formed in the absence of the blue dye.
  • Each of these test proteins also crystallized in the presence of dye, but crystallization of only four definitively resulted in blue crystals, specifically, lysozyme, tliauinatin, trypsin and equine seram albumin. Equine seram albumin displayed phase changes in the absence and presence of dye.
  • Crystallization of cellulase resulted in small flat square plates that appeared to display staining intensity, but it was not possible to definitively characterize the cellulase crystals as blue due to the thinness of the resultant crystals and the resulting similarity of the coloration to the background.
  • the color of cellulase crystals was similar to background hence it was not evident if these crystals absorbed the dye.
  • characterization of the cellulase crystals was further complicated by their small size.
  • the background intensity due to unincorporated dye present in each sample was similar for each protein tested.
  • the amount of dye incorporated into the protein crystals, and therefore the intensity of resulting coloration depended on the concentration of dye used and the number and size of the crystals ( Figure 1).
  • the blue dye Izit facilitated the screening of crystallization conditions suitable for producing crystals of lysozyme, pepsin and ⁇ -lactoglobulin.
  • Each ciystallization variable was represented by several implementations.
  • the variable organic precipitant was represented by five levels: 1, 2 ⁇ mefnyl-2,4-pentanediol (MPD); 2, polyethylene glycol 400 (PEG400); 3, PEG2000; 4, PEG4000; and 5, PEG8000.
  • the proteins used as test proteins for this example were: hen egg white lysozyme (Boehringer Mannheim, 1243004), 30 mg/ml in 0.1 M sodium acetate (pH 4.7); porcine pepsin (Sigma, P6887), 60 mg / ml in 100 rnM cacodylate buffer (pH 6.5), 0.2 M Ca acetate; bovine ⁇ -lactoglobulin (Sigma, L3908), 10 mg/ml in 100 mM Tris- HCL (pH 6.5).
  • the concentrations and buffer solutions used for the nano crystallization screen are identical to those used in the 0.5 ⁇ l scale experiments described earlier. Prior to the initiation of the nano crystallization screen IZIT was added to each sample using a dilution of 1 part dye to 19 parts protein (v/v).
  • MPD and ⁇ -OG are 2-methyl-2,4-pentanediol and N-octyl- ⁇ -d- gluocpyranoside.
  • PEG400, PEG2K, PEG4000 and PEG 8K are polyethylene glycols of indicated size/mass as is known to those of skill in the art.
  • the experimental nanoliter ciystallization volumes were sealed during the trials. Data were collected four days after the initiation of the experiment and recorded. The results were scored by visual inspection.
  • These categories include: (1) a clear drop, (2) phase separation, (3) precipitate (regular, granular), (4) microcrystals/precipitate, (5) rosettes or spheralites, (6) needles (one-dimensional growth), (7) plates (two-dimensional growth), (8) small crystals (three-dimensional growth of less than 0.2 mm) and (9) crystals (three- dimensional growth of greater than 0.2 mm).
  • the categories or scores used for the nanoliter crystallization trials were adopted from those suggested by Hampton Research. Scoring of individual experiments to assign each to a category was performed by visual inspection. As the titles of the categories suggest, the results of each experiment were scored based upon presence or absence of detectable species and upon the species' morphology.
  • needle-like crystals are those appear to have one axis that is very long compared to the others, while plates have two axes of approximately equivalent size, each significantly larger than the third.
  • a three dimensional crystal appears to have growth in three dimensions and looks like a brick.
  • precipitate looks like sand, while both phase separations and rosettes look like mixtures of oil and water.
  • the rosettes usually appear to have dense staining in their centers. In some instances, there is greater ambiguity as to the proper categorical assignment. In addition, the total number of trials in which blue crystals formed is also indicated.
  • the number of blue crystals is calculated from visual inspection of categories 6 - 9, i.e., needles, plates, small tliree dimensional crystals and larger three dimensional crystals.
  • a representative micrograph of blue crystals formed from each of the three proteins screened is shown in Figures 2-4.
  • Figure 2 shows a blue lysozyme crystal grown in the presence of Izit using screening condition 361
  • Figure 3 shows a blue pepsin crystal grown in the presence of Izit using screening condition 75
  • Figure 4 shows a blue ⁇ -lactoglobulin crystal grown in the presence of Izit using screening condition 213.
  • Condition 254 results not shown, resulted in no observable precipitation or crystallization.
  • Condition 251 ( Figure 5A) also does not result in any observable crystal formation.
  • condition 339 which includes acetate buffer at pH 4.5, 460 mM thiocyanate, 0.8% PEG 8000, 0.5% glycerol and 8 mM arginine-HCl, both result in apparent crystals.
  • Figure 5E and 5F show results from ⁇ -lactoglobulin under conditions with phosphate buffer at pH 8 and with PEG 400 (at Tl) and with phosphate buffer at pH 6, PEG 8000, 10% glycerol, 0.008M Arg-HCl (at T3), respectively.
  • Figure 5G, 5H, and 51 show results from myristol transferase under conditions 291 (at T3), 197 (at T2) and 259 (at T3), respectively.
  • Figure 5J shows results from pepsin under condition 347 (at T3).
  • Figures 5K and 5L show results from Renac2 under conditions 41 (at Tl) and 339 (at T3), respectively.
  • Figures 5M and 5N show results from Robl under conditions 212 (at T2) and 337 (at T3), respectively.
  • Figure 5O shows results from B10 in acetate buffer at pH 4.5, with 450 mM thiocyanate, 0.8% PEG 8000, 0.5% glycerol and 8 mM arginine HC1 (at T3).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Metallurgy (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Materials Engineering (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)
  • Crystals, And After-Treatments Of Crystals (AREA)

Abstract

L'invention concerne un procédé amélioré de criblage de conditions de croissance de cristaux, dans lequel des molécules sont cristallisées à partir de solutions contenant des colorants. Ces colorants sont sélectivement intégrés ou associés à des cristaux présentant un caractère particulier, pour conférer une coloration auxdits cristaux, et permettre ainsi une meilleure détection des cristaux colorés par colorant. Un procédé préféré consiste à utiliser des colorants dans des solutions protéiniques recouvertes d'huile. L'utilisation d'huile permet l'utilisation de petits volumes de solution et permet de faciliter le criblage d'un grand nombre de conditions de cristallisation dans des jeux ordonnés d'échantillons faisant appel à des dispositifs automatisés, permettant d'obtenir des solutions appropriées pour générer des essais de cristallisation, de recouvrir des essais de cristallisation avec une huile, de fournir des conditions appropriées conduisant à la cristallisation et d'améliorer la détection de cristaux colorés ou non colorés (par colorants) obtenus.
PCT/US2002/024032 2001-07-30 2002-07-30 Utilisation de colorant pour distinguer un sel et des cristaux de proteines dans des conditions de microcristallisation WO2003012430A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US30869801P 2001-07-30 2001-07-30
US60/308,698 2001-07-30
US32895801P 2001-10-12 2001-10-12
US60/328,958 2001-10-12

Publications (1)

Publication Number Publication Date
WO2003012430A1 true WO2003012430A1 (fr) 2003-02-13

Family

ID=26976398

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/024032 WO2003012430A1 (fr) 2001-07-30 2002-07-30 Utilisation de colorant pour distinguer un sel et des cristaux de proteines dans des conditions de microcristallisation

Country Status (1)

Country Link
WO (1) WO2003012430A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004005898A1 (fr) * 2002-07-10 2004-01-15 Uab Research Foundation Procede permettant de faire la distinction entre cristaux biomoleculaires et cristaux non biomoleculaires
EP1603068A3 (fr) * 2004-02-05 2006-02-22 RiNA Netzwerk RNA-Technologien GmbH Procédé pour surveiller la fabrication des cristaux des biomolécules
US7214540B2 (en) 1999-04-06 2007-05-08 Uab Research Foundation Method for screening crystallization conditions in solution crystal growth
US7244396B2 (en) 1999-04-06 2007-07-17 Uab Research Foundation Method for preparation of microarrays for screening of crystal growth conditions
US7247490B2 (en) 1999-04-06 2007-07-24 Uab Research Foundation Method for screening crystallization conditions in solution crystal growth

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4517048A (en) * 1983-10-31 1985-05-14 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Method for minimizing convection during crystal growth from solution
US5013531A (en) * 1990-08-31 1991-05-07 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Macromolecular crystal growing system
US6406903B2 (en) * 1995-09-25 2002-06-18 University Of Alabama At Birmingham Dynamically controlled crystal growth system
US6468346B2 (en) * 1999-12-10 2002-10-22 Bsi Proteomics Corporation Applying x-ray topography and diffractometry to improve protein crystal growth

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4517048A (en) * 1983-10-31 1985-05-14 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Method for minimizing convection during crystal growth from solution
US5013531A (en) * 1990-08-31 1991-05-07 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Macromolecular crystal growing system
US6406903B2 (en) * 1995-09-25 2002-06-18 University Of Alabama At Birmingham Dynamically controlled crystal growth system
US6468346B2 (en) * 1999-12-10 2002-10-22 Bsi Proteomics Corporation Applying x-ray topography and diffractometry to improve protein crystal growth

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7214540B2 (en) 1999-04-06 2007-05-08 Uab Research Foundation Method for screening crystallization conditions in solution crystal growth
US7244396B2 (en) 1999-04-06 2007-07-17 Uab Research Foundation Method for preparation of microarrays for screening of crystal growth conditions
US7247490B2 (en) 1999-04-06 2007-07-24 Uab Research Foundation Method for screening crystallization conditions in solution crystal growth
WO2004005898A1 (fr) * 2002-07-10 2004-01-15 Uab Research Foundation Procede permettant de faire la distinction entre cristaux biomoleculaires et cristaux non biomoleculaires
EP1603068A3 (fr) * 2004-02-05 2006-02-22 RiNA Netzwerk RNA-Technologien GmbH Procédé pour surveiller la fabrication des cristaux des biomolécules

Similar Documents

Publication Publication Date Title
US20070111317A1 (en) Use of dye to distinguish salt and protein crystals under microcrystallization conditions
Laurent et al. How do granitoid magmas mix with each other? Insights from textures, trace element and Sr–Nd isotopic composition of apatite and titanite from the Matok pluton (South Africa)
Chayen et al. Macromolecular crystallization and crystal perfection
Pusey et al. Life in the fast lane for protein crystallization and X-ray crystallography
Kamenetsky et al. Multiple mantle sources of continental magmatism: insights from “high-Ti” picrites of Karoo and other large igneous provinces
Santarsiero et al. An approach to rapid protein crystallization using nanodroplets
Tickle et al. High-throughput protein crystallography and drug discovery
Calero et al. Identifying, studying and making good use of macromolecular crystals
Martynowycz et al. Protocol for the use of focused ion-beam milling to prepare crystalline lamellae for microcrystal electron diffraction (MicroED)
Jin et al. Formation, chemical evolution and solidification of the dense liquid phase of calcium (bi) carbonate
Rakel et al. Moving through three‐dimensional phase diagrams of monoclonal antibodies
WO2003012430A1 (fr) Utilisation de colorant pour distinguer un sel et des cristaux de proteines dans des conditions de microcristallisation
Huang et al. Deep learning-aided high-throughput screening of time-resolved protein crystallization on programmable microliter-droplet systems
Blattmann et al. Growing crystals from protein
Snell et al. The application and use of chemical space mapping to interpret crystallization screening results
Mustafi et al. Near saturation of ribosomal L7/L12 binding sites with ternary complexes in slowly growing E. coli
AU2003266957A1 (en) Protein crystallisation method
Gorrec A beginner’s guide to macromolecular crystallization
Webster et al. Imaging structurally dynamic ribosomes with cryogenic electron microscopy
Lin et al. Transcription with a laser: Radiation-damage-free diffraction of RNA Polymerase II crystals
Cosenza et al. Use of dye to distinguish salt and protein crystals under microcrystallization conditions
Smithwick et al. Preparation of acid-fast microscopy smears for proficiency testing and quality control
Asanov et al. Intrinsic fluorescence as a potential rapid scoring tool for protein crystals
Barnard et al. Crystallization of integral membrane proteins
Duggins Confinement and Inclusions in Single Crystalline Biominerals

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG UZ VN YU ZA ZM

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载