U.S. patent application number 12/385012 was filed with the patent office on 2009-10-15 for device and method for forming macromolecule crystal.
Invention is credited to Koji Inaka, Tomoyuki Kobayashi, Moritoshi Motohara, Satoshi Sano, Masaru Sato, Shinichi Shinozaki, Sachiko Takahashi, Hiroaki Tanaka, Mari Yamanaka, Izumi Yoshizaki.
Application Number | 20090257929 12/385012 |
Document ID | / |
Family ID | 33487151 |
Filed Date | 2009-10-15 |
United States Patent
Application |
20090257929 |
Kind Code |
A1 |
Yoshizaki; Izumi ; et
al. |
October 15, 2009 |
Device and method for forming macromolecule crystal
Abstract
Disclosed is a macromolecule-crystal forming apparatus and
method capable of obtaining a macromolecule crystal in a simplified
and efficient manner. The device comprises a first container
containing a sample of macromolecule, a second container containing
a gel acting as a buffer material during the crystallization of the
macromolecule, and a third container containing a precipitant
solution having a function of facilitating the aggregation of
molecules during the crystallization of the macromolecule. These
containers are connected together in a given manner so as to allow
the macromolecule sample and the precipitant to be brought into
contact with one another through the gel to induce the
crystallization of the macromolecule.
Inventors: |
Yoshizaki; Izumi;
(Tsukuba-shi, JP) ; Sano; Satoshi; (Tsukuba-shi,
JP) ; Kobayashi; Tomoyuki; (Tsukuba-shi, JP) ;
Sato; Masaru; (Tsukuba-shi, JP) ; Motohara;
Moritoshi; (Tsukuba-shi, JP) ; Tanaka; Hiroaki;
(Shinjuku-ku, JP) ; Takahashi; Sachiko;
(Shinjuku-ku, JP) ; Shinozaki; Shinichi;
(Shinjuku-ku, JP) ; Yamanaka; Mari; (Shinjuku-ku,
JP) ; Inaka; Koji; (Yamatokoriyama-shi, JP) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W., SUITE 600
WASHINGTON
DC
20004
US
|
Family ID: |
33487151 |
Appl. No.: |
12/385012 |
Filed: |
March 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11287219 |
Nov 28, 2005 |
7531037 |
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12385012 |
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PCT/JP04/07682 |
May 27, 2004 |
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11287219 |
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Current U.S.
Class: |
422/187 |
Current CPC
Class: |
C30B 7/00 20130101; C30B
29/58 20130101; Y10T 117/1024 20150115; C30B 7/14 20130101; C30B
35/002 20130101; Y10T 117/10 20150115 |
Class at
Publication: |
422/187 |
International
Class: |
B01J 8/00 20060101
B01J008/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 27, 2003 |
JP |
2003-149585 |
Claims
1. A device for forming a macromolecule crystal, comprising: a
first container having at least one opening and containing a sample
of macromolecule; a second container having at least two openings
and containing a gel for buffering a crystallization solution
condition, a crystallization initiation period and a crystal-growth
rate of the macromolecule; and a third container having at least
one opening and containing a precipitant solution to be brought
into contact with the macromolecule sample in a liquid-liquid
diffusion process so as to facilitate the crystallization of the
macromolecule, wherein said first to third containers are arranged
to allow the macromolecule sample in said first container and the
precipitant solution in said third container to be diffused through
the gel in said second container, while allowing the precipitant
solution to be brought into contact with the macromolecule sample
within said first container.
2. The device as defined in claim 1, wherein the opening of said
first container is connected to a first one of the openings of said
second container, and a second one of said openings of said second
container is connected to the opening of said third container.
3. The device as defined in claim 2, wherein said second container
is made of an elastic material, wherein an end of said first
container having the opening thereof is inserted into one of the
ends of said second container each having a corresponding one of
the openings thereof, and an end of said third container having the
opening thereof is inserted into the other end of said second
container, whereby the connection between said respective ends is
maintained by means of the elastic force of said second
container.
4. The device as defined in claim 3, wherein said first and third
containers are connected to said second container in a displaceable
manner relative to one another.
5. A device for forming a macromolecule crystal, comprising: a
first container having at least one opening and containing a sample
of macromolecule; a second container having at least two openings
and containing a gel for buffering a crystallization solution
condition, a crystallization initiation period and a crystal-growth
rate of the macromolecule; and a third container incorporating said
first and second containers arranged in such a manner that the
opening of said first container and a first one of the openings of
said second container are connected together to allow the
macromolecule sample and the gel to be in contact with one another,
said third container containing a precipitant solution to be
brought into contact with the macromolecule sample via a
liquid-liquid diffusion process so as to facilitate the
crystallization of the macromolecule, wherein the gel and the
precipitant solution are in contact with one another at a second
one of said openings of said second container, so that the
precipitant solution is diffused into the macromolecule sample in
said first container through the gel.
6. The device as defined in claim 5, wherein said second container
is made of an elastic material, wherein an end of said first
container having the opening thereof is inserted into an end of
said second container having said first opening thereof, whereby
the connection between said ends is maintained by means of the
elastic force of said second container.
7. The device as defined in claim 6, wherein each of at least said
macromolecule sample and said precipitant solution is hermetically
sealed.
8. A device for forming a macromolecule crystal, comprising: a
first tubular container having an open end and containing a sample
of macromolecule; a second tubular container having first and
second opposite open ends and containing a gel for buffering a
crystallization solution condition, a crystallization initiation
period and a crystal-growth rate of the macromolecule; and a third
tubular container incorporating said first and second tubular
containers arranged in such a manner that the open end of said
first tubular container and the first open end of said second
tubular container are connected together to allow the macromolecule
sample and the gel to be in contact with one another, said third
tubular container containing a precipitant solution to be brought
into contact with the macromolecule sample via a liquid-liquid
diffusion process so as to facilitate the crystallization of the
macromolecule, wherein the precipitant solution is contained in
said third tubular container to allow the gel and the precipitant
solution to be in contact with one another at the second open end
of said second tubular container.
9. The device as defined in claim 8, wherein said first tubular
container is inserted into and connected to said second tubular
container, to establish the contact between the macromolecule
sample in said first tubular container and the gel in said second
tubular container.
10. The device as defined in claim 9, wherein said second tubular
container is designed to hold the open end of said first tubular
container received therein and maintain the connection therewith by
means of an elastic force.
11. The device as defined in claim 10, wherein said third tubular
container is formed in a test-tube-like shape having a lower
portion in which the precipitant solution is contained, wherein a
lower one of the open ends of said second tubular container is
immersed in the precipitant solution to establish the contact
between the precipitant solution and the gel in the second tubular
container so as to allow the precipitant solution to be diffused
into the gel and then into the macromolecule sample.
12. The device as defined in claim 11, wherein the macromolecule
sample in said first tubular container is diffused into the gel in
said second tubular container through the connected portion between
said first and second tubular containers, and the precipitant
solution is diffused into the gel in said second tubular container
and the macromolecule sample in said first tubular container
through an opening of the second open end of said second tubular
container.
13. The device as defined in claim 12, wherein each of said first
and third tubular containers is hermetically sealed.
14. The device as defined in claim 13, which is designed to induce
in said first tubular container a concentration gradient such that
the concentration of the macromolecule sample is increased in a
direction getting away from the interface between the gel in said
second tubular container and the macromolecule sample, and the
concentration of the precipitant solution is reduced in said
direction.
15-24. (canceled)
25. A device for forming a macromolecule crystal, comprising: a
first container having at least one opening and containing a sample
of macromolecule; a second container having at least two openings
and containing a porous material for buffering a crystallization
solution condition, a crystallization initiation period and a
crystal-growth rate of the macromolecule; and a third container
having at least one opening and containing a precipitant solution
to be brought into contact with the macromolecule sample via a
liquid-liquid diffusion process so as to facilitate the
crystallization of the macromolecule, wherein said first to third
containers are arranged to allow the macromolecule sample in said
first container and the precipitant solution in said third
container to be diffused through the porous material in said second
container, while allowing the precipitant solution to be brought
into contact with the macromolecule sample within said first
container.
26. A device for forming a macromolecule crystal, comprising: a
first container having at least one opening and containing a sample
of macromolecule; a second container having at least two openings
and containing a gel for buffering a crystallization solution
condition, a crystallization initiation period and a crystal-growth
rate of the macromolecule; and a plurality of third containers
connected with each other, each of said third containers having at
least one opening and containing a precipitant solution to be
brought into contact with the macromolecule sample via a
liquid-liquid diffusion process so as to facilitate the
crystallization of the macromolecule, wherein said first to third
containers are arranged to allow the macromolecule sample in said
first container and the precipitant solution in a selected one of
said third containers to be diffused through the gel in said second
container, while allowing the precipitant solution to be brought
into contact with the macromolecule sample within said first
container.
27. The device as defined in claim 26, wherein each of said third
containers has an opening for communication or connection with said
second container, and an additional opening provided with a
detachable sealing cap.
28-30. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to a device for
crystallization of macromolecules, and more particularly, to the
crystallization of macromolecules by a liquid-liquid diffusion
process.
BACKGROUND ART
[0002] In this day and age called the post-genomic era, instead of
traditional researches based on heuristic approach, a new approach
is required which is capable of developing biological
macromolecules with various structures on the basis of
comprehensive genome data, analyzing their molecular structures and
functions, artificially designing a molecular structure based on
the analytical result to prepare more highly functional
pharmaceutical molecules and proteins, and applying them to drug
discoveries, biological sciences, medical sciences or industries.
The acquisition of a higher-quality macromolecule crystal is
essential for performing the molecular structure analysis with a
higher degree of accuracy, and thus an experimental test on
crystallization of macromolecules is a very sort of basic and vital
step. What is firstly required under these circumstances is to
establish a technique for screening the conditions of a
crystallization test in a simplified and speedy manner and
preparing a high-quality crystal according to the obtained optimal
crystallization conditions, and the establishment of such a
technique is also a R & D that is societal demand. A primary
technique for crystallization of macromolecules includes a vapor
diffusion process, a batch process, a dialysis process and a
liquid-liquid diffusion process.
[0003] Heretofore, the vapor diffusion process has been most widely
used, because of relatively simple procedures of this process
itself, and the existence of a large amount of information about
crystallization accumulated in past years by many researchers and a
number of commercially available products and screening kits using
the vapor diffusion process.
[0004] However, optimal crystallization conditions for the vapor
diffusion process can be found only if a crystallization test is
repeatedly performed using a large number of combinations of
various macromolecule samples and various precipitating-agent or
precipitant solutions different in concentration. The reason comes
from the fact that, in the search of crystallization conditions for
the vapor diffusion process, the conditions of one sample drop can
be searched only in the linear interval between a certain initial
condition and a terminal condition determined by the concentration
of a precipitant solution in a reservoir. In contrast, the
crystallization process using liquid-liquid diffusion in a narrow
tube or tubule allows almost all concentration conditions to be
created at either point in a single tubule with time. Thus, it can
be said that the liquid-liquid diffusion process in a tubule is
simple and efficient as a technique for finding optimal
crystallization conditions.
[0005] In the conventional liquid-liquid diffusion process, the
crystallization of a macromolecule in a tubule has been performed
by forming layers of a macromolecule sample and a precipitant
solution in the tubule without mixing them up. In this technique,
the macromolecule sample and the precipitant solution are diffused
into one another through the interface therebetween. The
precipitant solution generally has a diffusion rate greater than
that of the macromolecule sample, and thereby the crystallization
will be initiated in a region of the macromolecule sample.
[0006] Late years, in this technique, there has been developed a
process of inducing the diffusion between the above two solutions
through a gel layer having a function of buffering the crystal
growth rate of a macromolecule. The use of this process can
eliminate the need for taking account of preventing intermixing
between the two solutions during formation of a double layer
thereof. This process also has a feature in that the intervention
of the gel allows the respective diffusion rates of the two
solutions to be reduced so as to provide an extended diffusion
time. It is desirable to induce the diffusion in sufficient time,
because a macromolecule crystal prepared at a lower growth rate
generally has a higher quality.
[0007] In the above conventional technique for forming a
macromolecule crystal by means of liquid-liquid diffusion to be
induced under the intervention of gel, a gel layer is formed in a
vessel, and a tubule containing a sample of macromolecule is put in
the vessel in such a manner as to insert the front end of the
tubule into the gel layer by a given length. After the insertion, a
precipitant solution is poured on the gel layer to form a double
layer structure of the gel layer and the precipitant solution
layer.
[0008] As time passes in this state, the precipitant solution in
the precipitant layer is diffused into the gel layer, and then
diffused from the front end of the tubule into the macromolecule
sample solution in the tubule. In this way, an intended environment
is created where the precipitant solution is brought into contact
with the macromolecule sample within the tubule to form a
macromolecule crystal. Then, as time further passes, the
precipitant solution is continuously diffused to have a
concentration gradient in which the concentration of the
precipitant solution is gradually reduced in a direction from the
front end toward the inside of the tubule. Concurrently, the
macromolecule sample in the tubule is reversely diffused into the
gel to have a concentration gradient in which the concentration of
the macromolecule sample in the tubule is gradually reduced in a
direction from the inside toward the front end of the tubule.
[0009] The presence of the above mutual concentration gradients
allows an optimal point for crystal growth of the macromolecule to
be provided with high probability so as to obtain an excellent
crystal.
[0010] However, it has been pointed out that the above conventional
crystal formation technique involves a problem about the risk of
outflow of the macromolecule sample in the tubule, technical
difficulties in inserting the tubule into the gel layer uniformly
by a given length, and variation in sample setting operation in a
case where it is required to prepare numbers and various types of
samples, resulting in poor quality of an obtained crystal or poor
process yield.
[0011] Further, the conventional technique cannot sufficiently meet
the need for preparing a great number of macromolecule sample
crystals in current researches.
[0012] Moreover, the device for the conventional technique involves
a structural problem about ineffective consumption of precipitant
and gel in large quantity.
DISCLOSURE OF INVENTION
[0013] In view of the above circumstances, it is therefore an
object of the present invention to provide a macromolecule-crystal
forming device and method capable of obtaining a macromolecule
crystal in a simplified and efficient manner.
[0014] It is another object of the present invention to provide a
macromolecule-crystal forming device and method capable of
obtaining an excellent or high-quality macromolecule crystal.
[0015] In order to achieve the above objects, according to a first
aspect of the present invention, there is provided a device for
forming a macromolecule crystal, which comprises a first container
having at least one opening and containing a sample of
macromolecule, a second container having at least two openings and
containing a gel for buffering the crystallization of the
macromolecule, and a third container having at least one opening
and containing a precipitant solution to be brought into contact
with the macromolecule sample so as to facilitate the
crystallization of the macromolecule, wherein the first to third
containers are arranged to allow the macromolecule sample in the
first container and the precipitant solution in the third container
to be diffused through the gel in the second container, while
allowing the precipitant solution to be brought into contact with
the macromolecule sample within the first container.
[0016] Preferably, the opening of the first container is connected
to a first one of the openings of the second container, and a
second one of the openings of the second container is connected to
the opening of the third container.
[0017] Preferably, the second container is made of an elastic
material, wherein an end of the first container having the opening
thereof is inserted into one of the ends of the second container
each having a corresponding one of the openings thereof, and an end
of the third container having the opening thereof is inserted into
the other end of the second container, whereby the connection
between the respective ends is maintained by means of the elastic
force of the second container.
[0018] Further, it is preferable that the first and third
containers are connected to the second container in a displaceable
manner relative to one another.
[0019] According to a second aspect of the present invention, there
is provided a device for forming a macromolecule crystal, which
comprises a first container having at least one opening and
containing a sample of macromolecule, a second container having at
least two openings and containing a gel for buffering the
crystallization of the macromolecule, and a third container
incorporating the first and second containers arranged in such a
manner that the opening of the first container and a first one of
the openings of the second container are connected together to
allow the macromolecule sample and the gel to be in contact with
one another, and containing a precipitant solution to be brought
into contact with the macromolecule sample so as to facilitate the
crystallization of the macromolecule, wherein the gel and the
precipitant solution are in contact with one another at a second
one of the openings of the second container, so that the
precipitant solution is diffused into the macromolecule sample in
the first container through the gel.
[0020] In this device, it is preferable that the second container
is made of an elastic material, and an end of the first container
having the opening thereof is inserted into an end of the second
container having the first opening thereof, whereby the connection
between the ends is maintained by means of the elastic force of the
second container.
[0021] Further, it is desirable that each of at least the
macromolecule sample and the precipitant solution is hermetically
sealed.
[0022] According to a third aspect of the present invention, there
is provided a device for forming a macromolecule crystal, which
comprises a first tubular container having an open end and
containing a sample of macromolecule, a second tubular container
having first and second opposite open ends and containing a gel for
buffering the crystallization of the macromolecule, and a third
tubular container incorporating the first and second tubular
containers arranged in such a manner that the open end of the first
tubular container and the first open end of the second tubular
container are connected together to allow the macromolecule sample
and the gel to be in contact with one another, and containing a
precipitant solution to be brought into contact with the
macromolecule sample so as to facilitate the crystallization of the
macromolecule, wherein the precipitant solution is contained in the
third tubular container to allow the gel and the precipitant
solution to be in contact with one another at the second open end
of the second tubular container.
[0023] In a preferred embodiment, the first tubular container is
inserted into and connected to the second tubular container, to
establish the contact between the macromolecule sample in the first
tubular container and the gel in the second tubular container.
[0024] Further, the second tubular container may be designed to
hold the open end of the first tubular container received therein
and maintain the connection therewith by means of an elastic
force.
[0025] In another preferred embodiment, the third tubular container
is formed in a test-tube-like shape having a lower portion in which
the precipitant solution is contained, wherein a lower one of the
open ends of the second tubular container is immersed in the
precipitant solution to establish the contact between the
precipitant solution and the gel in the second tubular container so
as to allow the precipitant solution to be diffused into the gel
and then into the macromolecule sample.
[0026] In still another preferred embodiment, the macromolecule
sample in the first tubular container is diffused into the gel in
the second tubular container through the connected portion between
the first and second tubular containers, and the precipitant
solution is diffused into the gel in the second tubular container
and the macromolecule sample in the first tubular container through
an opening of the second open end of the second tubular
container.
[0027] Each of the first and third tubular containers may have a
hermetically sealed end on the other side of their end connected to
the second tubular container. This makes it possible to prevent
vaporization of the macromolecule sample and the precipitant
solution so as to maintain adequate controllability.
[0028] In yet another preferred embodiment, the device is designed
to induce in the first tubular container a concentration gradient
such that the concentration of the macromolecule sample is
increased in a direction getting away from the interface between
the gel in the second tubular container and the macromolecule
sample, and the concentration of the precipitant solution is
reduced in the direction.
[0029] According to a fourth aspect of the present invention, there
is provided a method for forming a macromolecule crystal, which
comprises the steps of providing a first container having at least
one opening and containing a sample of macromolecule, providing a
second container having at least two openings and containing a gel
for buffering the crystallization of the macromolecule, connecting
the first and second containers through the opening of the first
container and a first one of the openings of the second container
to allow the macromolecule sample and the gel to be brought into
contact with one another, providing a third container containing a
precipitant solution to be brought into contact with the
macromolecule sample so as to facilitate the crystallization of the
macromolecule, and allowing the gel in the second container to be
brought into contact with the precipitant solution through a second
one of the openings of the second container while maintaining the
contact between the macromolecule sample in the first container and
the gel in the second container.
[0030] In one preferred embodiment, the method further includes the
steps of providing an elongated tube material which has opposite
open ends, supplying the macromolecule sample from one of the open
ends of the tube material to the inside of the tube material, and
cutting the tube material to a given length to form the first
container.
[0031] In this case, it is preferable to connect one of the open
ends of the tube material to a storage tank containing the
macromolecule sample, and connect a vacuum pump to the other open
end to form a negative pressure in the inside of the tube material,
or utilize a capillary phenomenon, so as to allow the macromolecule
sample to be supplied to the inside of the tube material.
[0032] Alternatively, a pump filled with the macromolecule sample
and connected to one of the open ends of the tube material may be
activated to supply the macromolecule sample to the inside of the
tube material.
[0033] In another preferred embodiment, the method further includes
the steps of providing an elongated tube material which has
opposite open ends, supplying the gel from one of the open ends of
the tube material to the inside of the tube material, and cutting
the tube material to a given length to form the second
container.
[0034] In this case, it is preferable to connect one of the open
ends of the tube material to a storage tank containing the gel, and
connect a vacuum pump to the other open end to form a negative
pressure in the inside of the tube material, so that the gel is
supplied to the inside of the tube material according to the
negative pressure.
[0035] Alternatively, a pump filled with the gel and connected to
one of the open ends of the tube material may be activated to
supply the gel to the inside of the tube material.
[0036] In these embodiments, the tube material may have
elasticity.
[0037] As a substitute for the gel, any suitable buffer material,
such as a porous material, having substantially the same function
as that of the gel, may be used.
[0038] According to a fifth aspect of the present invention, there
is provided a device for forming a macromolecule crystal, which
comprises a first container having at least one opening and
containing a sample of macromolecule, a second container having at
least two openings and containing a gel for buffering a
crystallization solution condition, a crystallization initiation
period and a crystal-growth rate of the macromolecule, and a
plurality of connected third containers each having at least one
opening and containing a precipitant solution to be brought into
contact with the macromolecule sample so as to facilitate the
crystallization of the macromolecule, wherein the first to third
containers are arranged to allow the macromolecule sample in the
first container and the precipitant solution in a selected one of
the third containers to be diffused through the gel in the second
container, while allowing the precipitant solution to be brought
into contact with the macromolecule sample within the first
container.
[0039] In one preferred embodiment, each of the third containers
has an opening for communication or connection with the second
container, and an additional opening provided with a detachable
sealing cap.
[0040] According to a sixth aspect of the present invention, there
is provided a method for forming a macromolecule crystal, which
comprises the steps of providing a first container having at least
one opening and containing a sample of macromolecule, providing a
second container having at least two openings and containing a gel
for buffering the crystallization of the macromolecule, connecting
the first and second containers through the opening of the first
container and a first one of the openings of the second container
to allow the macromolecule sample and the gel to be brought into
contact with one another, providing a plurality of integrally
connected third containers each containing a precipitant solution
to be brought into contact with the macromolecule sample so as to
facilitate the crystallization of the macromolecule, and allowing
the gel in the second container to be brought into contact with the
precipitant solution through a second one of the openings of the
second container while maintaining the contact between the
macromolecule sample in the first container and the gel in the
second container.
[0041] In this case, it is preferable to form each of the third
containers to have an opening for communication or connection with
the second container and an additional opening provided with a
detachable sealing cap, and perform the adjustment and replacement
of the precipitant solution through the additional opening.
[0042] According to a seventh aspect of the present invention,
there is provided a method for forming a macromolecule crystal,
which comprises the steps of providing a first container having at
least one opening and containing a sample of macromolecule,
providing a second container having at least two openings and
containing a gel for buffering the crystallization of the
macromolecule, connecting the first and second containers through
the opening of the first container and a first one of the openings
of the second container to allow the macromolecule sample and the
gel to be brought into contact with one another, providing a
plurality of integrally connected third containers each containing
a precipitant solution to be brought into contact with the
macromolecule sample so as to facilitate the crystallization of the
macromolecule, allowing the gel in the second container to be
brought into contact with the precipitant solution through a second
one of the openings of the second container while maintaining the
contact between the macromolecule sample in the first container and
the gel in the second container, and, after a lapse of a given time
from the initiation of the contact between the gel and the
precipitant solution, replacing a part or all of the precipitant
solution.
[0043] In this case, each of the third containers may have one end
located on the opposite side of the other end in contact with the
gel and formed with a through-hole, to allow the replacement of the
precipitant solution to be performed through the through-hole.
During the process of the crystallization, this through-hole is
appropriately sealed. When a portion of the third container having
the through-hole is made of a polymeric material, the sealing is
typically performed by a thermal fusion bonding process.
Alternatively, the through-hole may be sealed using a plug or
cap.
[0044] A better understanding of the present invention can be
obtained when the following detailed description of the preferred
embodiment is considered in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0045] FIG. 1 is a conceptual diagram of a macromolecule-crystal
forming device 10 of the present invention.
[0046] FIG. 2 is a schematic structural diagram showing a
macromolecule-crystal forming device 10 according to one embodiment
of the present invention.
[0047] FIG. 3 is an explanatory diagram of a process for forming a
silicon tube 5.
[0048] FIG. 4 is an external view showing a capillary 4 joined to
the silicon tube 5.
[0049] FIG. 5 is a graph showing a time-based variation in the
respective concentrations of lysozyme used as a protein, and sodium
chloride used as a precipitant.
[0050] FIG. 6 is a photomicrograph of a formed lysozyme
crystal.
[0051] FIG. 7 is a graph showing a time-based variation in the
respective concentrations of alpha amylase used as a protein, and
polyethylene glycol used as a precipitant.
[0052] FIG. 8 is a photomicrograph of a formed alpha amylase
crystal.
[0053] FIG. 9 is a schematic structural diagram showing a
macromolecule-crystal forming device according to another
embodiment of the present invention.
[0054] FIG. 10 is a schematic structural diagram showing a
macromolecule-crystal forming device according to yet another
embodiment of the present invention.
[0055] FIG. 11 is a schematic structural diagram showing a
macromolecule-crystal forming device according to still another
embodiment of the present invention.
[0056] FIG. 12 is a schematic structural diagram showing a
macromolecule-crystal forming device according to yet still another
embodiment of the present invention.
[0057] FIG. 13 is a schematic structural diagram showing a
macromolecule-crystal forming device according to another further
embodiment of the present invention.
[0058] FIG. 14 is a perspective view showing a syringe case unit 20
of a macromolecule-crystal forming device according to still a
further embodiment of the present invention.
[0059] FIG. 15 is a sectional view showing one of
macromolecule-crystal forming cells 10 incorporated in the syringe
case unit 20.
[0060] FIG. 16(a) is a top plan view of the syringe case unit
20.
[0061] FIG. 16(b) is a sectional view of the syringe case unit
20.
[0062] FIG. 17(a) is a sectional view of a cap 25.
[0063] FIG. 17(b) is a top plan view of the cap 25.
[0064] FIG. 18 is a perspective view showing the state after two of
the syringe case units 20 are stored in a storage box in a partly
superimposed manner.
[0065] FIG. 19 is a sectional view showing one of
macromolecule-crystal forming cells 10 incorporated in a syringe
case unit 20 of a macromolecule-crystal forming device according to
an additional embodiment of the present invention.
[0066] FIG. 20 is a sectional view of a cap 40.
[0067] FIG. 21 is a sectional view of a bush 30.
[0068] FIG. 22 is a photomicrograph of a formed lysozyme
crystal.
[0069] FIG. 23 is a graph showing a time-based variation in the
respective concentrations of a protein and a precipitant, wherein
the concentration of the precipitant is changed after a lapse of a
given time from the initiation of operation of the
macromolecule-crystal forming device in FIG. 19.
BEST MODE FOR CARRYING OUT THE INVENTION
[0070] With reference to the drawings, an embodiment of the present
invention will now be described.
[0071] Referring to FIG. 1, a macromolecule-crystal forming device
10 according to one embodiment of the present invention is
conceptually illustrated that is intended to form a crystal of a
protein using a solution containing the protein, which serves as a
macromolecule sample.
[0072] The macromolecule-crystal forming device 10 according to
this embodiment comprises a tubule-shaped first container or
capillary 4 which contains the macromolecule sample (protein
solution) 1, and a tubule-shaped first container or silicon tube 5
which contains a gel 3. The silicon tube 5 has opposite open ends,
and a cylindrical inner space filled with the gel in advance. The
gel 3 serves as a buffer material acting as a buffer to a
crystallization solution condition, a crystallization initiation
period and a crystal-growth rate in the process of a
crystallization phenomenon of the macromolecule. The
macromolecule-crystal forming device 10 further includes a third
container 7 containing a precipitant solution 2 having a function
of aggregating protein molecules during the crystallization of the
protein 1 to facilitate the crystallization.
[0073] One of the features of the present invention is to provide a
first container containing a sample solution of macromolecule, a
second container containing a gel acting as a buffer material
during the crystallization of the biomaterial, and a third
container containing a precipitant solution having a function of
facilitating the aggregation of molecules during the
crystallization of the macromolecule, and connecting these
containers in a given manner so as to allow the macromolecule
sample and the precipitant to be brought into contact with one
another through the gel to induce the crystallization of the
macromolecule. In this case, a gel and a precipitant solution may
be mixed together in the inner space of the second container to
form a gelatinized precipitant solution, and the obtained gelled
precipitant solution may be brought into contact with the
macromolecule sample in the first container.
[0074] With reference to FIG. 2 and the rest of the figures,
various specific embodiments will be described below.
[0075] Firstly, a tubule or capillary 4 containing a sample of
macromolecule (protein) is prepared. The macromolecule sample
(protein solution) used in this embodiment is shown in Table 1. The
capillary 4 in this embodiment is made of glass (length: 11 cm,
inner diameter: 0.3 mm), and the protein solution is contained in a
tubular inner space thereof. In this case, a given amount of
protein solution is sucked in the capillary 4, and then one of the
open ends of the capillary is sealed.
[0076] Then, a gel material [Agarose-III (product name), available
from Wako Pure Chemical Industries, Ltd.] is prepared.
[0077] The gel material is boiled and dissolved in water or
appropriate buffer solution to form a gel solution. Then, in this
embodiment, a single long silicon tube 5 is prepared, and one of
the open ends of the silicon tube 5 is immersed in a vessel 8
containing the gel solution, as shown in FIG. 3. A syringe 9 (made
of polypropylene) is attached to the other open ends of the silicon
tube 5, and then a plunger of the syringe 9 is pulled to induce a
negative pressure in an inner space of the silicon tube 5. Thus,
the inner space of the silicon tube 5 is filled with the gel
solution. Then, the gel solution is cooled in the inner space of
the silicon tube 5 to form the silicon tube 5 filled with a gel
3.
[0078] In a process of assembling a macromolecule-crystal forming
device 10, the gel-filled silicon tube 5 prepared in the above way
is cut to a given length (about 15 mm). One of the ends of the
silicon tube 5 cut to the given length is fitted onto the open end
of the capillary 4 to connect them together, as shown in FIG. 4.
This operation is performed while adequately taking account of
preventing air bubbles from entering between the silicon tube and
the tubule. If any air bubble enters therebetween, the gel-filled
silicon tube is pulled out once, and re-fitted. When the gel-filled
silicon tube 5 is adequately fitted onto the capillary 4, an
interface between the protein solution and the gel is formed at the
inserted end of the capillary 4. At the same time, a part of the
solidified gel is pushed out of the other end of the silicon tube
5. The pushed-out gel is cut off with a cutter.
[0079] The silicon tube 5 has elasticity which allows the
connection with the capillary 4 inserted into one end of the
silicon tube 5 to be elastically maintained. The capillary 4
connected with the silicon tube 5 is set up to a third container or
test tube 7 containing a precipitant solution 2. In this
embodiment, a 15 ml volume, capped test tube (diameter: about 16
mm, length: about 130 mm, material of the tube: glass, material of
the cap: melamine) is used as the test tube 7. About 3 ml of
precipitant solution 2 is poured in the test tube 7, and then the
capillary 4 connected with the cut silicon tube 5 is inserted into
the test tube 7.
[0080] In this operation, the silicon tube 5 connected with the
capillary 4 is incorporated into the test tube 7 in such a manner
that the other end or open end of the silicon tube 5 is immersed in
the precipitant solution 2. Thus, the gel is in contact with the
precipitant solution 2 on the side of the open end of the silicon
tube 5. The end of the capillary 4 in non-contact with the gel is
hermetically sealed by a seal 11. The seal 11 may be formed using
clay or grease. Alternatively, the seal 11 may be formed by
thermally sealing the end of the capillary 4. Further, the open end
of the test tube 7 is hermetically closed by a cap or plug 12.
[0081] This structure can prevent the vaporization of components of
the protein solution 1 and the precipitant solution 2 to maintain
their compositions homogeneously.
[0082] Table 1 shows the compositions of the protein solution and
the precipitant solution. A crystallization temperature in each
case is a room temperature.
TABLE-US-00001 TABLE 1 lysozyme Taka-amylase protein 100 mg/ml hen
egg white 90 mg/ml Taka-amylase solution lysozyme solution/50 mM
solution/50 mM acetate acetate buffer pH 4.5 buffer pH 6.0
precipitant 20% (w/v) sodium chloride/ 40% (w/v) polyethylene
solution 50 mM acetate buffer pH 4.5 glycol solution/50 mM acetate
buffer pH 6.0) crystallization room temperature room temperature
temperature
[0083] FIG. 5 shows the simulation-based estimated result of a
time-based variation in the respective concentrations of a protein
and a precipitant, wherein the simulation is performed on the
assumption that the macromolecule-crystal forming device 10
illustrated in FIG. 2 is used, lysozyme and sodium chloride being
used, respectively, as the protein for forming a macromolecule
sample or protein solution 1 and the precipitant for forming a
precipitant solution 2, and the concentrations being measured along
the longitudinal direction of the capillary 4 and the silicon tube
5. FIG. 6 is a photomicrograph of an actually formed crystal. In
FIG. 5, 100 mg/ml of the lysozyme indicates a concentration at the
edge of the capillary 4 fairly remote from the interface between
the lysozyme of the protein solution 1 and the gel 3. Further, 20%
of the precipitant solution indicates a concentration at the edge
of the inner space of the silicon tube 5 on the side remote from
the above interface. After about 6 hours from the setup of the
crystal forming device, the deposition of a crystal was initiated
at the edge of the capillary 4. Subsequently, within 24 hours, the
initiation of crystallization was observed in the entire region of
the capillary 4.
[0084] As with FIG. 5, FIG. 7 shows the simulation-based estimated
result of a time-based variation in the respective concentrations
of Taka-amylase used as a protein for forming a protein solution 1
and polyethylene glycol used as a precipitant for forming a
precipitant solution 2, in the inner space of the capillary 4 along
the longitudinal direction of the capillary 4 and the silicon tube
5. FIG. 8 is a photomicrograph of an actually formed crystal. In
this case, after several days from the setup of the
macromolecule-crystal forming device 10 having the structure in
FIG. 2, the initiation of crystallization was observed in the inner
space of the capillary 4.
[0085] According to the present invention, a macromolecule-crystal
forming device having a desired gel and precipitant solution can be
obtained quickly and readily in conformity to each of a plurality
of capillaries 4 containing various macromolecule samples. The gel
layer can also be supplied in a simplified and speedy manner to
achieve enhanced efficiency of an experimental test on
crystallization of macromolecules using a tubule.
[0086] In addition, the use of a gel-filled silicon tube allows the
volume of the gel to be reduced. Thus, the diffusion of the
precipitant solution into the tubule is accelerated to reduce a
time-period until the crystallization is initiated, or a
crystallization initiation period, as compared with a recently
developed crystallization method using a gel layer. This is
effective, particularly, in a case of using a precipitant solution
having a low diffusion rate.
[0087] Further, a crystal can be formed with significantly high
quality.
[0088] The respective concentrations of protein and precipitant
solutions required for forming at least one crystal can be
calculated in accordance with the estimated result of a time-based
variation in the respective concentrations of the solutions, and
the position and time of crystal formation in the capillary, and
the calculation result can be used to optimize crystallization
conditions.
[0089] A time-period required for completing the crystallization
can also be calculated.
[0090] Various other crystal forming devices usable in the present
invention will be described below.
[0091] An embodiment illustrated in FIG. 9 comprises a plurality of
capillaries 4, a plurality of curved silicon tubes 5 each attached
to the front end of the corresponding capillary 4, and a container
7 containing a precipitant solution, which is composed of a
multi-cell container, such as a type commonly used in a
crystallization test based on a vapor diffusion process. After a
precipitant solution is supplied into each of the cells, and the
tubules 4 each containing a protein solution are inserted into the
corresponding silicon tubes 5 each containing a gel 3, the lower
end of each of the silicon tubes 5 is immersed in the corresponding
cell. According to this embodiment, various crystallization tests
can be performed at the same time in a simplified manner.
[0092] In another embodiment illustrated in FIG. 10, a capillary 4
containing a protein solution is inserted into one of the ends of a
silicon tube 5 containing a gel 3, and a tubular container 7 filled
with a precipitant solution 2 in a liquid or gel form is connected
to the other end of the silicon tube 5. In this structure, the
silicon tube 5 is designed to be elastically deformable. Thus, the
relative positional relationship between the capillary 4 and the
precipitant container 7 can be advantageously adjusted freely.
[0093] In yet another embodiment illustrated in FIG. 11, a
precipitant-solution container 7 and a gel container 3 are
integrated together. Specifically, the container 7 is formed with a
passage 5a filled with a gel in advance to serve as a gel
container, and a precipitant reservoir in fluid communication with
the passage 5a and adapted to reserve a precipitant solution 2. A
capillary 4 filled with a protein solution 1 is inserted into and
connected to one of the open ends of the passage 5a constituting a
part of the container 7. In this manner, the contact between the
gel and the precipitant is established through the other open end
of the passage 5a in fluid communication with the precipitant
solution reservoir.
[0094] In still another embodiment illustrated in FIG. 12, as with
the embodiment illustrated in FIG. 11, a passage 5a is formed in a
part of a container 7 having a precipitant solution reservoir, and
a gel is filled and contained in the passage 5a. Then, a capillary
4 filled with a protein solution 1 is connected to a silicon tube 5
filled with a gel 3 as described in connection with FIG. 2, and the
silicon tube 5 is attached to the outer end of the passage 5a, or
gel-filled portion, of the container 7.
[0095] In yet still another embodiment illustrated in FIG. 13, a
gelatinized precipitant solution 6 prepared by mixing a gel and a
precipitant solution is filled in a silicon tube 5, and a capillary
4 containing a protein solution 1 is inserted into the silicon tube
5.
[0096] With reference to FIGS. 14 to 20, another further embodiment
of the present invention will be described.
[0097] In this embodiment, a macromolecule-crystal forming device
10 comprises a plurality of macromolecule-crystal forming cells
integrally connected with each other. Specifically, in this
embodiment, the macromolecule-crystal forming device 10 includes a
syringe case unit 20 formed by integrally connecting the plurality
of precipitant solution-containing containers 7 as described in the
aforementioned embodiments. This embodiment is characterized in
that the plurality of precipitant solution-containing containers or
syringe cases 21 are integrated together to form the syringe case
unit 20 having a unique configuration. FIG. 14 is a perspective
view of the syringe case unit 20. This embodiment has six
macromolecule-crystal forming cells integrally connected with each
other. Further, the syringe case unit 20 in this embodiment has six
cylindrical syringe cases 21 each having a given length, and these
cylindrical syringe cases 21 are arranged in parallel at given
intervals. Referring to FIG. 15, one of the macromolecule-crystal
forming cells incorporated in the syringe case unit 20 is
illustrated in sectional view.
[0098] The cell includes a capillary 4 filled with a protein
solution 1. In this embodiment, as a substitute for the silicon
tube 5, a polyimide tube 22 is used as a second container
containing a gel. This allows the container to be further reduced
in size. One of the ends of the capillary 4 is inserted into one of
the ends of the polyimide tube 22 containing a gel. The other end
of the capillary 4 on the opposite side of the connected portion
between the capillary 4 and the polyimide tube 22 is hermetically
sealed by grease 23. The assembled unit of the capillary 4 and the
polyimide tube 22 is fitted into each of the syringe cases 21
through a PVC tube 24 covering over the outer surface of the
polyimide tube 22. Each of the syringe cases 21 is filled with a
precipitant solution 1. In this embodiment, each of the syringe
cases 21 has a cylindrical shape with an opening at each of the
first and second opposite ends thereof. The opening of the first
end is closed by the assembled unit of the capillary 4 filled with
the protein solution 1 and the polyimide tube 22 filled with the
gel, which is inserted thereinto through the PVC tube 24, and the
opening of the second end is closed by a cap 25. Additionally
referring to FIGS. 16(a), 16(b), 17(a) and 17(b), FIGS. 16(a) and
16(b) are a top plan view and a sectional view of the syringe case
unit 20, respectively. As seen in these figures, the syringe cases
21 are integrally formed with a backing plate 27 to allow the
adjacent syringe cases 21 to be arranged at a constant interval.
This backing plate 27 has a guide portion 28 for guiding the
assembled unit of the capillary 4 and the polyimide tube 22 during
an operation of inserting the assembled unit into each of the
syringe cases 21. The guide portion 28 is formed by extending the
plate 27 beyond the first ends of the syringe cases 21. The
interval or distance between the adjacent syringe cases 21 is set
at a constant value slightly greater than the outer diameter of
each of the syringe cases 21. As shown in FIGS. 17(a) and 17(b),
the cap 25 has a convex portion 26 to be inserted into a
corresponding one of the cells, and six of the convex portions 26
are arranged at intervals corresponding to the cells. These caps 25
are integrally formed in a single piece in such a manner that the
six convex portions are located in opposed relation to the
corresponding openings of the six syringe cases 21.
[0099] In this embodiment, the capillary 4 has a length of 55 mm,
an inner diameter of 0.5 mm and an outer diameter of 1.25 mm. The
polyimide tube has a length of 12 mm, and the PVC tube has a length
of 5 mm.
[0100] A process of assembling the macromolecule-crystal forming
device 10 to be formed by integrally connecting the six cells,
according to the above embodiment, will be described below. In
advance of assembling, a gel is filled in the polyimide tube (in
the same manner as that in the aforementioned embodiments). The
convex portions of the caps 25 are fitted into the corresponding
openings in the second ends of the syringe cases 21 of the syringe
case unit 20 to hermetically seal the openings, and then a
precipitant solution is filled in the syringe cases 21. Then, a
polyimide tube filled with a gel is cut to a given length (12 mm in
this embodiment as described above). A PVC tube is also cut to a
given length (5 mm). Grease is applied to one end, or first end, of
a capillary to make ready to hermetically seal the first end of the
capillary 4 (in this stage, the first end is maintained in an open
state). The PVC tube 24 is fitted onto the other end, or second
end, of the capillary 4. A protein solution is sucked into and
filled in the capillary 4. Then, the first end of the capillary 4
is hermetically sealed by high vacuum grease. Then, the polyimide
tube 22 filled with the gel and cut to the given length is inserted
into the second end, or the non-grease-sealed end, of the capillary
4. Through this operation, the protein solution in the capillary 4
is brought into contact with the gel in the polyimide tube 22.
Then, the PVC tube 24 is slidingly moved to cover the outer surface
of the polyimide tube 22. In this state, the assembled unit of the
capillary 4 and the polyimide tube 22 is inserted into each of the
syringe cases 21. During this operation, the assembled unit of the
capillary 4 and the polyimide tube 22 can be slidingly inserted
into the opening of the syringe case 21 by use of the guide portion
28 formed in the syringe case unit, to provide enhanced efficiency
and reliability in the insertion operation. The polyimide tube 22
used in this process has an outer diameter of 1.2 mm and a wall
thickness of 0.006 mm, and the PVC tube 24 used in this process has
an outer diameter of 2 mm and a wall thickness of 0.5 mm. As
mentioned above, each of these tubes is prepared by cutting a
single long tube to a given length.
[0101] Referring to FIG. 18, two of the syringe case units 20 each
incorporating the assembled unit of the capillary 4 and the
polyimide tube 22 are located in face-to-face contact with one
another in a partially superimposed manner, as illustrated, to
allow them to be stored in a space-saving manner. As mentioned
above, the interval between adjacent syringe cases 21 is set at a
value slightly greater than the outer diameter of each of the
syringe cases 21. Thus, each of the syringe cases 21 of one of the
syringe case units 20 can be located within a corresponding one of
the intervals of the other syringe case unit 20. Thus, two of the
syringe case units 20 each incorporating a given number of complete
macromolecule-crystal forming cells can be disposed opposed to one
another in such a manner that the edge of each of the capillaries
of one of the syringe case units is located between the adjacent
syringe cases 21 of the other syringe case unit, and the respective
backing plates 27 are located outward, so as to allow the two
syringe case units 20 to be stored within a space having a length
of two times of that of the syringe case 21. In this manner, a
number of cells can be stored in a space-saving manner. Thus, in a
crystallization test to be performed under circumstances having
extremely hard spatial restrictions, for example a protein-crystal
forming test in outer space, the macromolecule-crystal forming
device according to this embodiment has an extremely valuable
advantage in allowing various tests to be performed with
significantly enhanced efficiency.
[0102] In this embodiment, two of the syringe case units 20
reversely oriented in the longitudinal and vertical directions and
partially superimposed on one another are stored in a single
storage box 8, as described above. As shown in FIG. 18, in this
embodiment, one syringe case unit 20 having six cells is stored in
a body portion 8a of the storage box 8, and the other syringe case
unit 20 having five cells is stored in a cover portion 8b of the
storage box 8.
[0103] Referring to FIGS. 19 to 21, a macromolecule-crystal forming
device 10 according to still a further embodiment of the present
invention is illustrated.
[0104] In this embodiment, as a substitute for the polyimide tube
22 in the foregoing embodiment, a second container to be filled
with a gel is composed of a bush 30 having three cylindrical
portions different in outer diameter, and two cylindrical portions
different in inner diameter. That is, the bush 30 has a large
outer-diameter portion 31, an intermediate outer-diameter portion
32, and a small outer-diameter portion 33. The large outer-diameter
portion 31 and the intermediate outer-diameter portion 32 have a
large inner-diameter portion, and the small outer-diameter portion
33 corresponds to a small inner-diameter portion. While each of the
caps 25 in the foregoing embodiment has the convex portion 26
corresponding to the opening of the syringe case, each of caps 40
in this embodiment 40 has a fit portion 41 protruding in such a
manner as to be inserted into the opening, a protruded portion 42
extending in a direction opposite to the fit portion 41, and a
communication hole 43 providing fluid communication between the
respective inner spaces of the protruded portion 42 and the fit
portion 41, as shown in FIG. 20.
[0105] As with the caps 25 in the foregoing embodiment, the unit of
the caps 40 in this embodiment is formed with notches 44 for
allowing the caps to be readily detached from the corresponding
cells individually.
[0106] A process of assembling the macromolecule-crystal forming
device according to this embodiment will be described below. A gel
is filled in the bush 30 in advance. A precipitant solution is
filled in each of the syringe cases 21, and the caps 40 are fitted
into the openings of the second ends of the corresponding syringe
cases 21. Then, a protein solution is filled in the capillary 4.
Then, in the same manner as that in the foregoing embodiment,
grease is applied to one end, or first end, of a capillary 4. The
first end of the capillary 4 is hermetically sealed by high vacuum
grease. The capillary 4 is inserted into the bush 30 to integrate
the capillary 4 filled with the protein solution with the bush 30
filled with the gel. This assembled unit is inserted from the side
of the small outer-diameter portion 33 of the bush 30 into each of
the syringe cases 21. As the result of this operation, a part of
the precipitant solution in the syringe cases 21 is pushed out from
openings 43 of the caps 40. In this operation, during the course of
inserting the assembled unit of the capillary 4 filled with the
protein solution and the bush 30 filled with the gel from the side
of the bush 30, the inner space of the syringe case is compressed
from the first end thereof to press the precipitant solution
therein. Thus, it is required to assure an escape route for the
precipitant solution. In this embodiment, the presence of the
opening 43 formed in each of the caps 40 to provide fluid
communication between the inner space of each of the syringe cases
21 and the outside makes it possible to adequately discharge an
excessive part of the precipitant solution through the opening 43
in response to the insertion of the assembled unit into the inner
space of the syringe case. Thus, the assembled unit of the
capillary 4 and the bush 30 can be inserted into each of the
syringe cases 21 while adequately maintaining the filling condition
of the precipitant solution in the syringe case. Then, the opening
43 of each of the protruded portion 42 is thermally sealed. In this
state, crystallization is promoted.
[0107] Table 2 shows test conditions using the device according to
this embodiment, wherein lysozyme and sodium chloride are used as
the protein solution and the precipitant solution,
respectively.
TABLE-US-00002 TABLE 2 lysozyme protein 120 mg/ml hen egg white
lysozyme/100 mM acetate solution buffer pH 4.5 (10 .mu.L)
precipitant 10% (w/v) sodium chloride/100 mM acetate buffer pH
solution 4.5 (150 .mu.L) crystallization room temperature (293 K)
temperature test result After 4 to 6 days from the start of a
crystallization test, crystal formation is initiated at 2 to 5 mm
from the lower end of the capillary.
[0108] FIG. 22 is a photomicrograph showing a lysozyme crystal
formed using the device according to this embodiment.
[0109] Referring to FIG. 23, a graph is illustrated in a chart form
that shows a time-based variation in the respective concentrations
of the protein solution and the precipitant solution, in the same
manner as that in FIGS. 5 and 7. That is, the graph shows the
variation in the respective concentrations of the protein solution
and the precipitant solution in the longitudinal direction of the
capillary 4 and the bush 30 filled with the gel. The vertical axis
represents a parameter indicative of the concentration of the
protein solution, and the horizontal axis represents a parameter
indicative of the concentration of the precipitant solution. In
FIG. 23, it is observed that the characteristic curves converge at
a position where the precipitant solution has a concentration
parameter of zero, and the protein solution has a concentration
parameter of 20. This means that the position corresponds to a
portion of the protein solution located on the side of the sealed
end 23 of the capillary. It is also observed that the
characteristic curves converge at a position where the precipitant
solution has a concentration parameter of 30, and the protein
solution has a concentration parameter of zero. This means that the
position corresponds to the connected point between the precipitant
solution portion 21 and the gel portion 30.
[0110] When a precipitant solution having a concentration parameter
of 30 in FIG. 23 is used, it is difficult to allow each of the
concentrations of protein and precipitant solutions to have a value
close to their initial concentration even after a lapse of long
times. Further, it is likely that a certain protein can be
crystallized only in such concentration conditions of these
solutions. In this case, the conventional device has great
difficulties in providing crystallization conditions for such a
protein. Through various researches in view of this circumstance,
the inventors found that crystallization conditions for such a
protein can be provided by use of the cap 40 with an opening to be
attached to the rear end of the syringe case 21. Specifically, the
inventors were designed to allow the precipitant solution to be
changed or replaced through the opening 43 in FIG. 20 so as to
provide wider crystallization conditions. More specifically, a
process of replacing a part or all of the precipitant solution
after a lapse of a given time from the initiation of the contact
between the gel and the precipitant solution in the above device,
or after the initiation of the crystallization phenomenon of a
protein, is additionally provided. In this case, a third container
or syringe case 21 is formed with a through-hole at the second end
on the other side of the first end in contact with the gel, and the
cap 40 attached to this through-hole is detached to perform the
replacement of the precipitant solution. After the cap 40 is
attached again, the through-hole 43 will be appropriately sealed if
the crystallization phenomenon is induced. When a portion of the
third container having the through-hole is made of a polymeric
material, the sealing is typically performed through a thermal
fusion bonding process. Alternatively, the through-hole may be
hermetically sealed by use of a plug.
[0111] For example, a macromolecule-crystal forming device having a
structure as shown in FIG. 19 is constructed by using a protein
solution having a given concentration and a precipitant solution
having a given concentration, and crystallization is promoted for a
given time-period (e.g. one week). Then, a high-concentration
precipitant solution is filled in the inner space of the syringe
case 21 through the cap 40 to allow the obtained precipitant
solution to have a concentration parameter of 60. When the
concentration of the precipitant solution is increased (a
concentration parameter is increased from 30 to 60 in this
embodiment) through the cap 40 after a lapse of a give time from
the initiation of the crystallization, a plurality of concentration
characteristic curves extending beyond the line of a
precipitation-solution concentration parameter of 30 are formed as
shown in FIG. 23. Thus, concentration conditions for both the
protein and precipitant solutions, which are hardly achieved by the
combinations of initial concentrations of the protein and
precipitant solutions, can be provided quickly and readily by use
of the macromolecule-crystal forming device having the structure in
FIG. 19.
INDUSTRIAL APPLICABILITY
[0112] As mentioned above, according to the present invention, a
macromolecule crystal can be formed using liquid-liquid diffusion
through a gel in a simplified and efficient manner.
[0113] In particular, according to the present invention, even in a
case where it is required to prepare numbers and various types of
samples, a macromolecule-crystal forming device having a desired
gel and precipitant solution corresponding to a desired
macromolecule solution can be obtained quickly and readily. In this
case, the gel layer can be supplied in a simplified and speedy
manner to achieve enhanced efficiency of an experimental test on
crystallization of macromolecules using a tubule.
[0114] In addition, the use of a gel-filled silicon tube in one
embodiment of the present invention allows the volume of the gel to
be reduced as compared to the conventional device. Thus, the
diffusion of the precipitant solution into the tubule can be
accelerated to reduce a crystallization initiation period, as
compared with the conventional crystallization method using a gel
layer. In an actual test on lysozyme, while crystallization in the
conventional method is initiated after about 7 days, the present
invention allows crystallization to be initiated after about 6
hours. In another actual test on Taka-amylase, while
crystallization in the conventional method is initiated after about
10 days, the present invention allows crystallization to be
initiated after about 3 days. Thus, the present invention is
effective, particularly, in a case of using a precipitant solution
having a low diffusion rate. Further, the present invention can
form a crystal with significantly high quality.
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