U.S. patent application number 10/465576 was filed with the patent office on 2003-12-25 for apparatus for crystal growth of biomacromolecules.
This patent application is currently assigned to Japan Atomic Energy Research Institute. Invention is credited to Arai, Shigeki, Chatake, Toshiyuki, Kurihara, Kazuo, Maeda, Mitsuru, Niimura, Nobuo, Onishi, Yuki.
Application Number | 20030233978 10/465576 |
Document ID | / |
Family ID | 29728303 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030233978 |
Kind Code |
A1 |
Niimura, Nobuo ; et
al. |
December 25, 2003 |
Apparatus for crystal growth of biomacromolecules
Abstract
A method comprising the steps of continuously changing the
concentrations in solution of a biomacromolecule to be crystallized
and a precipitant, thereby constructing a crystal phase diagram
containing a solubility curve, searching for optimum conditions of
crystallization on the basis of the constructed crystal phase
diagram, and performing efficient growth of the crystal of the
biomacromolecule. Also disclosed is an apparatus for implementing
the method.
Inventors: |
Niimura, Nobuo; (Ibaraki,
JP) ; Onishi, Yuki; (Ibaraki, JP) ; Arai,
Shigeki; (Ibaraki, JP) ; Chatake, Toshiyuki;
(Ibaraki, JP) ; Maeda, Mitsuru; (Ibaraki, JP)
; Kurihara, Kazuo; (Ibaraki, JP) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
Japan Atomic Energy Research
Institute
Kashiwa-shi
JP
|
Family ID: |
29728303 |
Appl. No.: |
10/465576 |
Filed: |
June 20, 2003 |
Current U.S.
Class: |
117/200 |
Current CPC
Class: |
Y10T 117/1012 20150115;
Y10T 117/10 20150115; C30B 7/00 20130101; C30B 29/58 20130101 |
Class at
Publication: |
117/200 |
International
Class: |
C30B 035/00; C30B
001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 21, 2002 |
JP |
2002-181988 |
Claims
What is claimed is:
1. An apparatus for the crystal growth of biomacromolecules which
crystallizes a biomacromolecule with a precipitant to yield the
crystal of the biomacromolecule, comprising: (i) a first
compartment for accommodating a first fluid containing a
biomacromolecule to be crystallized; (ii) a second compartment for
accommodating a second fluid containing a precipitant; (iii) a
concentration adjusting means for adjusting the concentration of
the biomacromolecule in said first fluid and the concentration of
the precipitant in said second fluid; and (iv) a detection means
for continuously detecting the crystal growth of said
biomacromolecule in the first fluid in said first compartment.
2. The apparatus for the crystal growth of biomacromolecules
according to claim 1, wherein said concentration adjusting means
has a dialysis means that is provided at the boundary between the
first and second compartments and which establishes solution
communication between the first and second fluids.
3. The apparatus for the crystal growth of biomacromolecules
according to claim 1, wherein said detection means is a visual
detection means and has a light transmitting window provided on the
first compartment such that light passes through the first
compartment to enable visual detection of the crystal growth in
it.
4. The apparatus for the crystal growth of biomacromolecules
according to claim 3, wherein said detection means has a polarizing
filter on the optical path to said light transmitting window.
5. The apparatus for the crystal growth of biomacromolecules
according to claim 4, wherein said detection means has a light
source and a detection unit for detecting the light issued from the
light source, the polarizing filter comprises a first polarizing
filter element that polarizes the light from the light source
before it illuminates the light transmitting window and a second
polarizing filter element that polarizes the light from the light
source after it passes through the light transmitting window to
travel to the outside of the first compartment, said detection
means being so constructed that it detects the light from the light
source after it has passed through the first polarizing filter
element to be launched into the light transmitting window on the
first compartment, through which it passes to emerge from the light
transmitting window and then passes through the second polarizing
filter element.
6. The apparatus for the crystal growth of biomacromolecules
according to claim 5, wherein said detection unit is an optical
microscope, a CCD or a digital camera.
7. The apparatus for the crystal growth of biomacromolecules
according to any one of claims 1-6, wherein the dialysis means is a
semipermeable membrane that is not permeable to the
biomacromolecule in the first fluid which is to be crystallized but
is permeable to the precipitant in the second fluid.
8. The apparatus for the crystal growth of biomacromolecules
according to any one of claims 1-7, which further includes a system
temperature control means.
9. A method of producing the crystal of a biomacromolecule using
the apparatus according to claim 1 for the crystal growth of
biomacromolecules, said method comprising the steps of detecting
the crystal growth of a biomacromolecule over time by the
aforementioned detection means to determine the correlation between
the concentration of a precipitant, the concentration of the
biomacromolecule and the grown crystal of the biomacromolecule,
representing the determined correlation in graphic form to
construct a basic crystal phase diagram, and growing the crystal of
the biomacromolecule under optimum crystallizing conditions on the
basis of the constructed crystal phase diagram.
10. A method in which the crystal of a biomacromolecule to be
crystallized is grown under optimum conditions by continuously
changing the concentration of the biomacromolecule and that of a
precipitant, said method comprising the steps of: (a) increasing
the concentration of the biomacromolecule and/or the concentration
of the precipitant so that the solution containing said
biomacromolecule is shifted to the supersaturated region of a phase
diagram for that biomacromolecule, thereby causing it to form
crystal nuclei; and (b) thereafter adjusting the concentration of
the biomacromolecule and/or that of the precipitant such that the
solution containing said biomacromolecule is shifted to the
metastable region of the phase diagram for that biomacromolecule,
thereby growing the crystal nuclei formed in step (a).
11. The method according to claim 10, which further includes step
(c) of choosing only one crystal nucleus between steps (a) and
(b).
12. The method according to claim 10 or 11, wherein the
biomacromolecule to be crystallized is a protein.
13. The method according to claim 10 or 11, wherein the
biomacromolecule to be crystallized is a nucleic acid.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to an apparatus for efficient
preparation of the crystals of biomacromolecules such as proteins
and nucleic acids. The invention also relates to a method for
preparing the crystals of biomacromolecules in which the
concentration in solution of a biomacromolecule such as a protein
or nucleic acid and the concentration of a precipitant are adjusted
on a real-time basis to construct a crystal phase diagram for the
biomacromolecule and, in addition, on the basis of the constructed
crystal phase diagram, the solution of the biomacromolecule is
shifted between a supersaturated and a metastable region to effect
nucleation and growth of the biomacromolecule's crystal.
[0002] Following the success in mapping of the human genome, an
international project has launched with the hope of determining the
three-dimensional structures (about 10,000 in number) of the
structural families of all proteins in five years. With a view to
determining 3,000 out of those 10,000 proteins primarily by x-ray
analysis and NMR, Japan has started the so-called Project 3000
Proteins. It is not clear how many of the 3,000 proteins are going
to be determined by x-ray analysis of crystal structures but 2,000
would be a minimum number.
[0003] In the x-ray analysis of crystal structures, a single
crystal to be subjected to structural analysis is essential and
preparing such a single crystal has been a bottleneck in the
art.
[0004] Vapor diffusion is one of the conventional methods commonly
employed to prepare single crystals. In this method, the moisture
in a solution that lies in the unsaturated region of a phase
diagram for the molecule to be crystallized is vaporized, whereby
the concentration of the molecule to be crystallized and that of a
precipitant are increased so that the state of the solution is
shifted to the supersaturated region in the phase diagram, thereby
starting the growth of a crystal and causing it to grow. In this
method of crystal growth, the concentration of the molecule to be
crystallized and that of the precipitant are not controlled
artificially, so in order to prepare a single crystal of a certain
molecule to be subjected to structural analysis, it has been
necessary to prepare solutions of the molecule under a huge number
of conditions and cause it to crystallize out of the individual
solutions.
[0005] In addition, as the solvent vaporizes, the concentration of
the molecule to be crystallized and that of the precipitant will
only change to increase, so once the solution is shifted into the
unsaturated region with a view to forming crystal nuclei, it is
difficult to readjust the conditions of the solution to enter the
metastable region which provides more preferred conditions for
crystal growth.
[0006] On account of these problems, single crystal preparation by
the vapor diffusion method has been considerably wasteful both
time-wise and money-wise.
[0007] In order to solve the above-mentioned problems with the
vapor diffusion method, a robot-assisted method for high-throughput
crystal growth has been devised (WO 01/92293; Wilding, P. and
Kricka, L. J., TIBTECH, 17, 465-468 (1999)). However, this is no
more than a proposal for handling protein solutions by a machine,
rather than humans, in the vapor diffusion method and it by no
means provides a new technique for crystal growth. As a matter of
fact, the problems with the vapor diffusion method are yet to be
solved by the proposal.
[0008] Even today, the essence of structural biology lies in
unraveling the physiological functions of a certain molecule at the
atomic level by determining the hydrogen atoms and structure of
hydration through high-resolution structural analysis and neutron
diffractometry, both being intended to attain a resolution in the
neighborhood of 1 angstrom. To meet this need, single crystals are
required that are either of good quality or larger than 1
mm.sup.3.
[0009] Single crystals that satisfy these requirements are
difficult to obtain by just repeating the conventional mechanical
trial-and-error approach and it is strongly desired to develop a
method and an apparatus that facilitate the production of
biomacromolecules as single crystals having the required level of
quality.
SUMMARY OF THE INVENTION
[0010] An object, therefore, of the present invention is to provide
an apparatus for efficient preparation of single-crystal
biomacromolecules.
[0011] Another object of the invention is to provide a method for
efficient preparation of single-crystal biomacromolecules.
[0012] The present inventors have undertaken intensive efforts to
develop a reasonable technique for crystal growth. As a result,
they discovered a method comprising the steps of continuously
changing the concentrations in solution of a biomacromolecule to be
crystallized and a precipitant, thereby constructing a crystal
phase diagram containing a solubility curve, searching for optimum
conditions of crystallization on the basis of the constructed
crystal phase diagram, and performing efficient growth of the
crystal of the biomacromolecule.
[0013] The inventors also constructed an apparatus capable of
implementing the method. The present invention has been
accomplished on the basis of these findings.
[0014] More specifically, the present invention provides an
apparatus for the crystal growth of biomacromolecules which
crystallizes a biomacromolecule with a precipitant to yield the
crystal of the biomacromolecule, comprising:
[0015] (i) a first compartment for accommodating a first fluid
containing a biomacromolecule to be crystallized;
[0016] (ii) a second compartment for accommodating a second fluid
containing a precipitant;
[0017] (iii) a concentration adjusting means for adjusting the
concentration of the biomacromolecule in said first fluid and the
concentration of the precipitant in said second fluid; and
[0018] (iv) a detection means for continuously detecting the
crystal growth of said biomacromolecule in the first fluid in said
first compartment.
[0019] Whenever the term "crystal growth apparatus" appears on the
following pages, it shall mean the apparatus for the crystal growth
of biomacromolecules.
[0020] The crystal growth apparatus of the invention which has the
structural design shown above fills the first compartment with the
first fluid containing the biomacromolecule to be crystallized and
achieves efficient growth of its crystal while continuously
monitoring the formation and growth of that crystal in the first
fluid.
[0021] The crystal growth apparatus of the invention may be so
designed that the concentration adjusting means has a dialysis
means that is provided at the boundary between the first and second
compartments and which establishes solution communication between
the first and second fluids.
[0022] The dialysis means is preferably formed of a semipermeable
membrane that is not permeable to the biomacromolecule in the first
fluid but is permeable to the precipitant in the second fluid and
the solvent used in the first and second fluids.
[0023] Suitable semipermeable membranes are those which are capable
of differentiating the biomacromolecule from the precipitant and
solvent by an appropriate index such as molecular weight and
specific examples are Microcon (Millipore Corporation, MA, USA) and
Molecular/Por (Spectrum Laboratories, CA, USA).
[0024] In the present invention, it is preferred to differentiate
the biomacromolecule from the precipitant and solvent by molecular
weight. The pore size of the semipermeable membrane used to
differentiate the biomacromolecule from the precipitant and solvent
by molecular weight can be easily determined by the skilled artisan
on the basis of the relationship between the molecular weight of
the biomacromolecule to be crystallized and that of the
precipitant.
[0025] Since the concentration adjusting means has the dialysis
means of the nature described above, the biomacromolecule in the
first fluid does not diffuse into the second fluid whereas the
precipitant in the second fluid can freely diffuse into the first
fluid and then back to the second fluid and the solvent in the
first and second fluids is also free to diffuse between the first
and second fluids. Thus, by changing the amount of the solvent in
the first fluid, the concentration of the biomacromolecule in the
first fluid can be adjusted appropriately while, at the same time,
the concentration of the precipitant in the second fluid can be
changed by causing it to migrate into the first fluid or by causing
mutual movements of the solvent in the first and second fluids.
[0026] It is also preferred in the present invention that the first
and second fluids establish communication only through the dialysis
means provided at the boundary between the first and second
compartments and that no communication of solutions is established
through any other means. The term "solution" as used herein means a
liquid having a solute dissolved in a solvent and if a precipitate
has occurred in a solution, only the part that is left after
removing the precipitate is regarded as "solution".
[0027] Because of the above-described characteristics of the
dialysis means, if a higher pressure is exerted on the first fluid,
the solvent and precipitant in the first fluid diffuse into the
second fluid and the volume of the first fluid decreases, whereupon
it has a higher concentration of the biomacromolecule. Conversely,
if a lower pressure is exerted on the first fluid, the solvent and
precipitant in the second fluid flow into the first fluid and the
volume of the first fluid increases, whereupon it has a lower
concentration of the biomacromolecule.
[0028] Therefore, in the present invention, the concentration
adjusting means preferably has a pressure adjusting means in the
first compartment in order to adjust the pressure on the first
fluid and change its volume, thereby adjusting the concentration of
the biomacromolecule in it. In this way, the concentration of the
biomacromolecule in the first fluid can be changed to a desired
value. Examples of the pressure adjusting means include but are not
limited to a syringe and bellows that are movable to perform
pressure control. In the invention, it is preferred to use a
syringe that is movable to perform pressure control.
[0029] If the concentration of the precipitant in the second fluid
is increased, the concentration of the precipitant in the first
fluid can be increased accordingly and if the concentration of the
precipitant in the second fluid is decreased, the concentration of
the precipitant in the first fluid can be decreased
accordingly.
[0030] Therefore, in the present invention, the concentration
adjusting means preferably has a precipitant feed/discharge means
on the second compartment by which the precipitant is fed into and
discharged from the second compartment in order to adjust its
concentration in the second fluid. As a result, the dialysis means
provided at the boundary between the first and second fluids
enables the precipitant to be fed into or discharged from the first
fluid. The precipitant feed/discharge means may be of any type that
can replace the second fluid in the second compartment by a
solution of either a higher or a lower concentration of the
precipitant. A suitable example is one or more pumps that are
capable of adding a solution to the second fluid or removing a
solution from it. In the present invention, it is preferred to use
one or more pumps that are capable of adding a solution to the
second fluid or removing a solution from it. Specifically, feed
pumps for use in liquid chromatography are suitable.
[0031] Thus, in the present invention, using the above-described
concentration adjusting means, one can selectively adjust either
the concentration of the biomacromolecule in the first fluid or the
concentration of the precipitant in the first and second fluids.
Alternatively, the concentration of the biomacromolecule in the
first fluid and that of the precipitant in the first and second
fluids may be adjusted simultaneously but independently of each
other.
[0032] The term "the first and second fluids" as used in the
invention refers conveniently to the fluids contained in the first
and second compartments, respectively. While both fluids are
variable in composition during crystal growth, we describe below
the compositions of the first and second fluids in the initial
state.
[0033] The first fluid in the initial state has the solvent and the
biomacromolecule as main ingredients, with other additives being
optionally contained as required. The concentration of the
biomacromolecule to be crystallized is chosen as appropriate for
its specific type.
[0034] The second fluid in the initial state has the solvent and
the precipitant as main ingredients, with other additives being
optionally contained as required. The precipitant is chosen by the
skilled artisan as appropriate for the type of the biomacromolecule
to be crystallized and its concentration may be altered as
appropriate for its specific type.
[0035] The biomacromolecule to be crystallized in the invention
encompasses proteins and nucleic acids but other substances are
also included if they are biomacromolecules that are intended to be
crystallized.
[0036] If nucleic acids are to be crystallized, a variety of
precipitants may be employed, including inorganic salts such as
ammonium sulfate ((NH.sub.4).sub.2SO.sub.4), potassium phosphate
(K.sub.3PO.sub.4), sodium phosphate (Na.sub.3PO.sub.4), calcium
chloride (CaCl.sub.2), magnesium chloride (MgCl.sub.2), sodium
chloride (NaCl), potassium chloride (KCl) and nickel chloride
(NiCl.sub.2), organic solvents such as 2-methyl-2,4-pentanediol
(MPD), acetone, ethanol, methanol, isopropanol, dioxane and
butanol, and poly(ethylene glycol). If proteins are to be
crystallized, a variety of precipitants may also be employed and
they include but are not limited to ammonium sulfate
((NH.sub.4).sub.2SO.sub.4- ) and sodium chloride (NaCl).
[0037] The solvent used in the first fluid is the same as the
solvent in the second fluid. Water may be mentioned as a specific
example.
[0038] The detection means in the crystal growth apparatus of the
invention is for continuously monitoring the growth of the crystal
of the biomacromolecule in the first fluid. When the term
"continuously" is used in the invention, it is not necessarily
required that monitoring be performed at all times and it may be
performed at regular intervals, preferably no longer than one day,
more preferably no longer than 12 hours, and most preferably no
longer than 1 hour.
[0039] The detection means as a component of the crystal growth
apparatus of the invention is preferably a visual detection means
and has a light transmitting window provided on the first
compartment such that light passes through the first compartment to
enable visual detection of the crystal growth in it. The detection
means is not limited to a visual type and may be a spectroscopic
detection means such as one relying on dynamic light
scattering.
[0040] Crystals often become transparent when they are in solution
and a simple attempt at visually monitoring the transmitted light
often fails to detect its entity fully. The crystal in solution can
be monitored as a phase difference image by using a polarizing
filter. Therefore, if the detection means is a visual type, a
polarizing filter is preferably provided on the optical path to the
light transmitting window.
[0041] In another preferred embodiment, the detection means has a
light source and a detection unit for detecting the light issued
from the light source, the polarizing filter comprises a first
polarizing filter element that polarizes the light from the light
source before it illuminates the light transmitting window and a
second polarizing filter element that polarizes the light from the
light source after it passes through the light transmitting window
to travel to the outside of the first compartment, said detection
means being so constructed that it detects the light from the light
source after it has passed through the first polarizing filter
element to be launched into the light transmitting window on the
first compartment, through which it passes to emerge from the light
transmitting window and then passes through the second polarizing
filter element.
[0042] Examples of the detection unit that may be used by the
detection means of the invention for the purpose of visually
monitoring the crystal growth of the biomacromolecule include
recording devices such as a camera and video recorder and observing
devices such as an optical microscope. Considering preferred
conditions such as capability for continuous monitoring and ease of
recording, recording devices such as a camera and a video recorder
that use a CCD or observing devices such as an optical microscope
are preferably used as the detection unit in the invention.
[0043] The crystal growth apparatus of the invention may further
comprise a system temperature control means for maintaining the
temperatures of the first and second fluids at desired levels so
that the conditions for crystal growth are held constant. In this
case, the temperatures of the first and second fluids are
preferably maintained at 4-30.degree. C., more preferably at
6-22.degree. C., and most preferably at 18-20.degree. C.
[0044] We next describe the method of producing the crystal of a
biomacromolecule.
[0045] According to the second aspect of the invention, there is
provided a method of producing the crystal of a biomacromolecule
using the above-described apparatus for the crystal growth of
biomacromolecules. The method comprises the steps of detecting the
crystal growth of a biomacromolecule over time by the
aforementioned detection means to determine the correlation between
the concentration of a precipitant, the concentration of the
biomacromolecule and the grown crystal of the biomacromolecule,
representing the determined correlation in graphic form to
construct a basic crystal phase diagram, and growing the crystal of
the biomacromolecule under optimum crystallizing conditions on the
basis of the constructed crystal phase diagram.
[0046] The term "phase diagram" as used herein refers to a diagram
showing a range in which a supersaturated and a undersaturated
region in a two-phase system are in thermodynamic equilibrium and
in which a supresaturated region and a unsaturated region are
divided by a solubility curve. Phase stable regions in phase
diagrams are generally expressed as a function of two variables
chosen from among temperature, concentration, pressure, etc. In the
case of the present invention, the concentration of the
biomacromolecule to be crystallized and that of the precipitant are
two such variables.
[0047] In a phase diagram for a two-phase system of the type
contemplated in the present invention which consists of a
supersaturated and a undersaturated region, the supersaturated
region and the undersaturated region which are separated by the
solubility curve are not the only regions that exist and the
supersaturated region consisted of two specific regions, a
"supersaturated region" in a narrow definition (hereinafter
referred to as "supersaturated region") where crystal nuclei are
formed and crystals can grow and a "metastable region" where
crystals grow but crystal nuclei are not formed. In the
"supersaturated region", crystal nuclei are formed one after
another, so if this region is maintained too long, the number of
crystal grains formed will inevitably increase and their size
decreases. On the other hand, if a desired number of crystal nuclei
are first formed in the "supersaturated region" and then a shift is
made to the "metastable region", the number of crystal grains can
be controlled to a desired value and they still have a desired
size.
[0048] If the method of the invention is performed using the
crystal growth apparatus of the invention, the concentration of the
biomacromolecule to be crystallized and that of the precipitant can
be controlled freely and independently of each other and it is free
to create a "supersaturated region" or a "metastable region" within
the first compartment. In order to know the conditions under which
a phase shift occurs, it has heretofore been necessary to conduct a
large number of experiments under various conditions and find the
conditions that allow for actual formation of crystals. According
to the present invention, one only need perform a single
crystallization to draw a solubility curve and construct a phase
diagram while simultaneously determining the "supersaturated
region" and the "metastable region". In other words, the apparatus
of the invention for crystal growth of biomacromolecules is capable
of not only causing crystal grains to grow but also constructing a
crystal phase diagram.
[0049] As described above, according to the invention, the
concentration of the biomacromolecule to be crystallized and that
of the precipitant can be controlled freely and independently of
each other. Therefore, the object of the invention can also be
attained by a method in which the crystal of a biomacromolecule to
be crystallized is grown under optimum conditions by continuously
changing the concentration of the biomacromolecule and that of a
precipitant, said method comprising the steps of:
[0050] (a) increasing the concentration of the biomacromolecule
and/or the concentration of the precipitant so that the solution
containing said biomacromolecule is shifted to the supersaturated
region of a phase diagram for that biomacromolecule, thereby
causing it to form crystal nuclei; and
[0051] (b) thereafter adjusting the concentration of the
biomacromolecule and/or that of the precipitant such that the
solution containing said biomacromolecule is shifted to the
metastable region of the phase diagram for that biomacromolecule,
thereby growing the crystal nuclei formed in step (a).
[0052] The term "adjusting the concentration" refers to both
increasing and decreasing the concentration. Therefore, in step
(b), the concentrations of the biomacromolecule and the precipitant
may both be increased or decreased depending on the case, freely
and independently of each other. By performing steps (a) and (b) in
appropriate ways, a desired number of crystal nuclei are formed in
the supersaturated region and then a shift is made to the
metastable region so that the number of crystal grains are
controlled to a desired value while adjusting them to a desired
size.
[0053] In an embodiment of the invention, step (c) of choosing only
one crystal nucleus may be included between steps (a) and (b).
[0054] In step (a), more than one crystal nucleus may be formed in
the solution after it is shifted to the supersaturated region. In
this case, by choosing the single largest nucleus, more efficient
crystal growth can be realized and, in addition, the
biomacromolecule present in a limited amount in the sample can be
intensively used to grow a single crystal.
[0055] In the invention, various methods can be employed to choose
only one of the crystal nuclei formed in the solution. In one
method, the concentration of the biomacromolecule and/or that of
the precipitant is adjusted such that the solution is shifted to
the unsaturated region and crystal nuclei are dissolved until only
one crystal nucleus remains in the solution. In another method,
only one crystal nucleus is left physically intact and all other
crystal nuclei are removed. In yet another method, only one crystal
nucleus is isolated from other crystal nuclei. In the invention, it
is preferred to adopt the method of adjusting the concentration of
the biomacromolecule and/or that of the precipitant such that the
solution is shifted to the unsaturated region and crystal nuclei
are dissolved until only one crystal nucleus remains in the
solution. In this method, the largest of the crystal nuclei that
are present in the solution will usually remain undissolved and the
solution containing only such residual crystal nuclei may be
re-shifted into the metastable region for starting another cycle of
crystal growth.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] FIG. 1 is a schematic diagram showing a preferred embodiment
of the present invention as it relates to an apparatus for crystal
growth of biomacromolecules;
[0057] FIG. 2 is a phase diagram showing how a solution containing
hen egg lysozyme turned out when the concentration of sodium
chloride was increased from 1% to 3%, with the concentration of hen
egg lysozyme kept constant;
[0058] FIG. 3 is a phase diagram showing how a solution containing
hen egg lysozyme turned out when the concentration of sodium
chloride was decreased from 3% to 1%, with the concentration of hen
egg lysozyme kept constant;
[0059] FIG. 4 shows by two micrographs the formation of
single-crystal grains of hen egg lysozyme in solution as the
concentration of sodium chloride was increased from 1% (left
picture) to 3% (right picture), with the concentration of hen egg
lysozyme kept constant;
[0060] FIG. 5 shows by two micrographs the redissolution of
single-crystal grains of hen egg lysozyme as the concentration of
sodium chloride was decreased from 3% (left picture) to 1% (right
picture), with the concentration of hen egg lysozyme kept
constant;
[0061] FIG. 6 is a phase diagram showing how a solution containing
hen egg lysozyme turned out when the concentration of sodium
chloride was increased from 1% to 2%, with the concentration of hen
egg lysozyme kept constant;
[0062] FIG. 7 is a phase diagram showing how a solution containing
hen egg lysozyme turned out when the concentration of hen egg
lysozyme was decreased from 59.0 mg/mL to 17.2 mg/mL, with the
concentration of sodium chloride maintained at 2%;
[0063] FIG. 8 shows by four micrographs the formation of
single-crystal grains of hen egg lysozyme in solution as the
concentration of sodium chloride was increased from 1% (top left
picture) to 2% (bottom right picture), with the concentration of
hen egg lysozyme kept constant; and
[0064] FIG. 9 shows by four micrographs the growth of crystals of
hen egg lysozyme in solution as the concentration of hen egg
lysozyme was decreased from 59.0 mg/mL (top left picture) to 17.2
mg/mL (bottom right picture), with the concentration of sodium
chloride maintained at 2%.
DETAILED DESCRIPTION OF THE INVENTION
[0065] One preferred embodiment of the invention is hereunder
described with reference to the accompanying drawings but it should
be understood that the invention is by no means limited to that
particular embodiment.
[0066] FIG. 1 is a schematic diagram showing a preferred embodiment
of the invention as it relates to an apparatus for crystal growth
of biomacromolecules. The apparatus generally indicated by 1 in
FIG. 1 is for crystallizing a biomacromolecule with a precipitant
and causing the crystal to grow. It comprises a first compartment
10 for a first fluid containing the biomacromolecule to be
crystallized, a second compartment 20 for a second fluid containing
the precipitant, a concentration adjusting means 30 for adjusting
the concentration of the biomacromolecule in the first fluid and
that of the precipitant in the second fluid, and a detection means
40 for continuously detecting the crystal growth of the
biomacromolecule in the first fluid in the first compartment.
[0067] To describe further, the concentration adjusting means 30
has a dialysis membrane 32 as a dialysis means that is provided at
the boundary between the first compartment 10 and the second
compartment 20 to establish solution communication between the
first and second fluids. In the embodiment under consideration, the
dialysis membrane 32 is a semipermeable membrane having the
characteristics already described above.
[0068] The first compartment 10 is in cylindrical form and has the
dialysis membrane 32 at an end. The first compartment 10 has at the
other end a syringe 31 that is part of the concentration adjusting
means 30 and which pressurizes or evacuates the interior of the
first compartment 10 so that the solvent in the first fluid in it
is diffused into the second compartment 20 or is drawn back into
the first compartment 10. Structural details of the syringe 31 are
not shown but it fits the inner surfaces of the first compartment
10 so tightly that the first fluid will not leak out.
[0069] The second compartment 20 is a hollow cylinder having a
constriction 21 in the center to be generally shaped like "H" in
cross-sectional form. It has a feed inlet 33a at the front end and
a discharge outlet 33b at the rear end. The solvent and precipitant
that are ingredients in the second fluid are fed into the second
compartment via the inlet 33a and discharged from the second
compartment via the outlet 33b. The feed inlet 33a and the
discharge outlet 33b are other components of the concentration
adjusting means 30. Thus, in the embodiment under consideration,
the concentration adjusting means 30 consists of the syringe 31,
dialysis membrane 32, feed inlet 33a and discharge outlet 33b. The
constriction 21 in the second compartment 20 has a slightly larger
inside diameter than the outside diameter of the first compartment
10.
[0070] The detection means 40 is a visual type and has a light
transmitting window 41 provided on the first compartment 10 in such
a way that light passes through the first compartment 10 to enable
visual detection of crystal growth in it. In the embodiment under
consideration, the peripheral wall of the first compartment 10 is
thoroughly formed of a light transmitting material, specifically,
clear quartz glass, so that the first compartment 10 is enclosed
with the light transmitting window 41 throughout. The constriction
21 of the second compartment 20 is also formed of a light
transmitting material, specifically, clear quartz glass. Hence, the
second compartment 20 is also fitted with a light transmitting
window 41'.
[0071] In the embodiment under consideration, the detection means
40 has a polarizing filter on the optical path to the light
transmitting windows 41 and 41'. It also has a light source 43 and
a detection unit 45 for detecting the light issued from the light
source 43, and the polarizing filter comprises a first polarizing
filter element 42a that polarizes the light from the light source
43 before it illuminates the light transmitting windows 41 and 41'
and a second polarizing filter element 42b that polarizes the light
from the light source 43 after it passes through the light
transmitting windows 41 and 41' to travel to the outside of the
first compartment 10. The detection means 40 is so constructed that
it detects the light from the light source 43 after it has passed
through the first polarizing filter element 42a to be launched into
the light transmitting window 41 on the first compartment 10,
through which it passes to emerge from the light transmitting
window 41 and then passes through the second polarizing filter
element 42b. In the embodiment under consideration, the first and
second polarizing filter elements 42a and 42b are both provided on
the outer surfaces of the second compartment 20.
[0072] In the embodiment under consideration, a mirror 44 is
provided so that the light passing through the second polarizing
filter element 42b is guided into a CCD camera as the detection
unit 45.
[0073] In the embodiment under consideration, a heater and an
insulation jacket are provided (not shown) as the system
temperature control means. In the embodiment under consideration,
the temperatures of the first and second fluids are controlled at
6-22.degree. C. by such temperature control means.
[0074] Using the above-described apparatus for the crystal growth
of biomacromolecules, the invention detects the crystal growth of a
biomacromolecule over time by the aforementioned detection means to
determine the correlation between the concentration of a
precipitant, the concentration of the biomacromolecule and the
grown crystal of the biomacromolecule, represents the determined
correlation in graphic form to construct a basic crystal phase
diagram, and grows the crystal of the biomacromolecule under
optimum crystallizing conditions on the basis of the constructed
crystal phase diagram.
[0075] In this method, the biomacromolecule's crystals of a desired
size can be efficiently obtained by (a) increasing the
concentration of the biomacromolecule and/or the concentration of
the precipitant so that the solution containing the
biomacromolecule is shifted to the supersaturated region of a phase
diagram for that biomacromolecule, thereby causing it to form
crystal nuclei and (b) thereafter adjusting the concentration of
the biomacromolecule and/or the concentration of the precipitant so
that the solution containing the biomacromolecule is shifted to the
metastable region of the phase diagram for that biomacromolecule,
thereby growing the crystal nuclei formed in step (a).
[0076] The present invention is by no means limited to the
foregoing embodiment and various improvements and modifications can
be made without departing from the spirit and scope of the
invention.
[0077] For example, two cylindrical vessels having the same
diameter may be connected with a semipermeable membrane interposed
in such a way as to prevent the leakage of the internal fluids. One
of the two cylindrical vessels is used as the first compartment and
the other used as the second compartment. After being filled with
the first fluid, the first compartment is fitted with the syringe
described above; on the other hand, the second compartment is
provided with the feed inlet and the discharge outlet and then
filled with the second fluid. In this case, the visual detection
means has the same construction as the one described in the
foregoing embodiment, except that a light transmitting window is
provided on the first compartment but not on the second
compartment.
[0078] The following examples are provided for the purpose of
further illustrating the present invention but are in no way to be
taken as limiting.
EXAMPLE 1
[0079] Changing the Concentration of Precipitant and Crystal
Formation
[0080] Hen egg lysozyme (59.0 mg/mL) in a buffer solution (50 mM
sodium acetate/acetic acid, pH 4.7) was used as a biomacromolecule
to be crystallized. Using sodium chloride in a buffer solution (50
mM sodium acetate/acetic acid, pH 4.7) as a precipitant, the hen
egg lysozyme was crystallized in a thermostatic incubator at
22.degree. C. Spectra/Por MWCO=3500 (Spectrum Laboratories, CA,
USA) was used as a dialysis membrane. A 10-.mu.L micro-dialysis
button (Hampton Research, CA, USA) was used as the first
compartment and a 50-.mu.L screw vial was used as the second
compartment.
[0081] In this example, the reaction system was left to stand for
26 hours with the concentration of hen egg lysozyme in the buffer
solution being kept constant but the concentration of sodium
chloride increased from the initial 0% to 1%. Then, the
concentration of sodium chloride as the precipitant was further
increased to 3% and the reaction system was left to stand for 19
hours. Thereafter, the concentration of sodium chloride was
reverted to 1% and the reaction system was left to stand for 20
hours. The changes in the concentration of sodium chloride are
depicted in the phase diagrams in FIGS. 2 and 3. The temperatures
of the first and second fluids were controlled with an incubator
(MIR153 of Sanyo Electric Co., Ltd.)
[0082] With the concentration of sodium chloride being varied, the
state of crystallization of the hen egg lysozyme in solution was
observed with a microscope (SZX of OLYMPUS OPTICAL CO., LTD.,
Tokyo), a CCD camera (KP-C251 of Hitachi Electronics Co., Ltd.) and
a computer (Macintosh Power PC 7600 of Apple Computer Inc., CA,
USA). As it turned out, crystals formed with increasing
concentration of the precipitant (FIG. 4) and they dissolved again
into solution with decreasing concentration of the precipitant
(FIG. 5).
EXAMPLE 2
[0083] Changing the Concentrations of Precipitant and
Biomacromolecule and Crystal Formation
[0084] In this example, hen egg lysozyme was crystallized under the
same conditions as in Example 1, except that the concentrations of
hen egg lysozyme as biomacromolecule and sodium chloride as
precipitant were changed continuously under different
conditions.
[0085] First, the reaction system was left to stand for 7 days with
the concentration of hen egg lysozyme being held constant but the
concentration of sodium chloride adjusted to the initial 1%.
Thereafter, the concentration of sodium chloride as the precipitant
was increased to 2%. Then, the concentration of sodium chloride in
solution was maintained at a constant level of 2%. The
concentration of hen egg lysozyme decreased from the initial 59.0
mg/mL to 17.2 mg/mL over 16 days. The changes in the concentrations
of sodium chloride and hen egg lysozyme are depicted in the phase
diagrams in FIGS. 6 and 7, respectively.
[0086] With the concentration of sodium chloride or hen egg
lysozyme being varied as described above, the state of
crystallization of hen egg lysozyme in solution was observed using
the same devices as those employed in Example 1. As it turned out,
crystals formed with increasing concentration of the precipitant
(FIG. 8) and the crystal nuclei formed under that condition grew
with the lapse of time when the concentration of the
biomacromolecule dropped from 59.0 mg/mL to 17.2 mg/mL (FIG.
9).
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