Automated biopolymer crystal preparation apparatus

Preparation of biopolymer crystals is made automatic by an apparatus comprising a reactor system, a solution liquid supply system, temperature control system, and a control unit controlling the supply system and the temperature control system, and preferably, an image recording system.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to an apparatus automatically preparing 
crystals of a biopolymer such as proteins and nucleic acids in accordance 
with a predetermined procedure. 
2. Description of the Related Art 
The atomic level structure determination of proteins by X-ray analysis has 
been made in molecular biology, protein engineering, etc., and solving the 
crystal structure of a portion helps in understanding the relationship 
between the three-dimensional molecular structure or atomic arrangement of 
the main chain of the amino acid and the enzyme action thereof. 
In a crystal structure analysis of a protein, (1) it is necessary to 
determine the optimum conditions for preparing crystals of the protein 
with a very small amount of protein sample, and (2) a certain size (about 
several hundred micrometers) of a crystal is necessary for X-ray 
diffraction. Many factors have an influence on the crystallization of a 
protein, including the concentration of a protein, the kind and 
concentration of a neutral salt and an organic solvent, pH, temperature, 
and contaminants, etc. To obtain large and satisfactory crystals, the 
crystallization of a protein under zero gravity in space has been 
considered and tried. 
Heretofore, the preparation of biopolymer crystals has been done manually 
by skilled persons in accordance with their skill and experience. But 
considerable time and work are necessary to determine the optimum 
conditions for crystal preparation, since there are many factors such as 
the concentration of a biopolymer, the kind and concentration of a 
biopolymer insolubilizing agent, the ionic strength, pH, and temperature 
of a reaction solution, etc. Further, in manual operation, the results may 
be varied by slight differences in procedure, resulting in an undesirable 
low reproducibility. 
K. Nakamura and Y. Mitsui in "Crystallization of Protein by Hanging-drop 
Method" Rigaku Denki Journal 16, 1985, pp 12-14 describe a hanging-drop 
method in which a vessel for tissue culture having a plurality of 
reservoirs is used so that the crystallization conditions of a protein are 
examined systematically. 
In this operation, a sample solution and ammonium sulfate solution are 
dropped on a coverglass by means of a digital micropipette and combined by 
means of a fine glass rod. The coverglass is then placed upside down on a 
reservoir, and this operation is repeated for all reservoirs. This 
operation is disadvantageous in that, if the operation is not done 
quickly, water evaporation changes the concentration of the solution. 
Other known apparatuses for preparing biopolymer crystals include that 
disclosed by, for example, Walter Littke and Christina John in "Protein 
Single Crystal Growth Under Microgravity", SCIENCE, Vol. 225, pp 203-204; 
and Yuhei Moriguchi in "Crystal Growth of Proteins under Microgravity", 
"Nihon Kessho Gakkai Shi (Japanese Journal of Crystallography)" 28, 47 
(1986) pp 47-49. All of these apparatuses were developed for use in space, 
but were not intended to provide an automated apparatus. Further, an 
apparatus for preparing biopolymer crystals in a land-based laboratory has 
not been proposed. 
SUMMARY OF THE INVENTION 
The object of the present invention is to provide an automated apparatus 
for preparing biopolymer crystals, in which the optimum conditions for 
crystal preparation are automatically determined and reproducibility of 
the biopolymer crystals is improved. 
The above and other objects, features, and advantages are attained by an 
automated apparatus for preparing biopolymer crystals, comprising: (a) a 
reactor system in which biopolymer crystals are grown; (b) a supply system 
comprising reservoirs, pipes, a pump, and switch valves for supplying 
reaction solutions to the reactor system; (c) means for controlling the 
temperature in the reactor system; and (d) means for controlling the 
supply system and the temperature controlling means in a predetermined 
procedure for conducting the preparation of biopolymer crystals in the 
reactor system. Preferably, the apparatus further comprises a system for 
recording the process of biopolymer crystal growth in the reactor, and the 
reactor system preferably comprises a plurality of reactors. 
According to the present invention, various process and conditions for 
preparing various crystals can be established or selected as desired 
through the supply system including the switch valves; a biopolymer 
solution and biopolymer insolubilizing agent solution can be supplied to 
the reactor system in desired concentration and flow rate; a plurality of 
crystal preparations can be conducted concurrently by supplying necessary 
solutions to each reactor of the plurality of reactors forming the reactor 
system.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
An example of an automated apparatus for preparing biopolymer crystals 
according to the present invention is described with reference to FIGS. 1 
to 9. 
FIG. 1 is a block diagram of the apparatus according to the present 
invention. In the apparatus, under the control of a central control system 
1, reaction solutions are sent from a supply system 2 to a reactor system 
5, the temperature of which is controlled by a temperature control system 
4, and a process of crystallization in the reactor system 5 is recorded by 
an image recording system 7. 
FIG. 2 is a more detailed construction of the apparatus shown in FIG. 1. 
The supply system 2, in this case, comprises four solution reservoirs 21 
to 24 for a biopolymer solution, a biopolymer insolubilizing solution, a 
buffer, and a wash solution, reaction solution-selecting switch valves 25 
and 26, stirrers 27 and 28, a first pump 29, a second pump 30, a liquid 
passage-selecting switch valve 31, three cooperating twelve-way rotary 
valves 32 to 34, a waste reservoir 35, and a supply system control device 
20 which controls the reaction solution-selecting switch valves 25 and 26, 
the stirrers 27 and 28, the pumps 29 and 30, the passage-selecting switch 
valve 31, and the twelve-way rotary valves 32, 33 and 34. In FIG. 2, the 
four solution reservoirs 21 to 24 may have a cap or they may be a closed 
system, if desired. 
FIGS. 3A and 3B are perspective and sectional views of the reactor system 
5, respectively. The reactor system 5 comprises eleven reactors 51 to 61 
and pipes 51a to 61a, 51b to 61b and 51c to 61c connecting between each of 
the reactors 51 to 61 and each of the three twelve-way rotary valves 32 to 
34. In FIG. 2, only one set of the pipes 51a, 51b, and 51c is shown for 
simplicity. 
Each reactor comprises an inner peripheral vessel A, an outer peripheral 
vessel B, and a connecting part C connecting the inner and outer 
peripheral vessels A and B. The connecting part C has a structure by which 
the liquids in the inner and outer peripheral vessels A and B can be kept 
separate but vapor diffusion allowed therebetween. Preferably, the 
reactors are made of a transparent material such as glass or plastic to 
allow the state of the crystal growth to be recorded by the image 
recording system 7. 
The pipes 51a to 61a are connected to the twelve-way rotary valve 32, the 
pipes 51b to 61b are connected to the twelve-way rotary valve 33, and the 
pipes 51c to 61c are connected to the twelve-way rotary valve 34, 
respectively. 
FIG. 3A illustrates the reactor system 5 which is composed of eleven 
reactors 51 to 61 and is in the shape of a doughnut. 
FIG. 3B illustrates the connections between the respective reactors and 
twelve-way rotary valves. 
In FIG. 2, the temperature control system 4 comprises a heat accumulating 
chamber 41, a stirrer 42, a heater 43, a heat sensor 44, and a control 
device 40 in the temperature control system 4, which device controls the 
heater 43 and the stirrer 42 by signals from the heat sensor 44. In this 
example, the reactors 51 to 61 are surrounded by or included in the heat 
accumulating chamber 41. In order to carry out image recording, preferably 
at least a part of the heat accumulating chamber 41 used in an image 
recording optical system is transparent. 
The image recording system 7 comprises an illuminating light source 71, a 
light-passage switching mirror and a driving unit 72 thereof, a TV camera 
73, a video tape recorder 74, and a control device 70 in the image 
recording system which device controls the light source 71, a unit of the 
mirrors and the drive 72 thereof, the TV camera 73, and the video tape 
recorder 74, etc. 
FIGS. 4A and 4B illustrate a more detailed construction of the unit of the 
light passage switching mirror and the drive 72 thereof. FIG. 4A is a 
plane view and FIG. 4B is a sectional view, in which light from the light 
source 71 passes through a heat absorbing filter 75 and a scattered light 
filter 76, then passes through a desired reactor, is reflected by an 
optical pass switching mirror 72a, and reaches the TV camera to project 
images of the biopolymer crystals therein. The optical pass switching 
mirror 72a enables the observation and image recording of the state of 
crystal growth in the desired reactor, by rotation or vertical movement 
made by a driving unit 72b. 
As seen in FIG. 4B, reactors may be provided at multiple levels, as shown 
by reactors 5 and 5'. In such a case, the optical pass switching mirror 
72a is moved downward when recording at the lower reactor 5' is to be 
done. The TV camera 73 is preferably movable. 
In FIG. 2, the supply-system control device 20, the temperature 
control-system control device 40 and the image recording-system control 
device 70 are controlled by a central control device 1. 
Examples for preparing biopolymer single crystals by (1) a batch-wise 
method; (2) a vapor diffusion method; and, (3) a free interface diffusion 
method, using the automated apparatus for preparing biopolymer crystals 
shown in FIG. 2, are described below. 
The operation of the pumps 29 and 30 and valve 31 and the state of a 
reaction solution in a reactor in the above three kinds of methods for 
preparing biopolymer crystals are shown in FIG. 5. As seen in FIG. 5, in 
the batch-wise method, a mixture of biopolymer and insolubilizing 
solutions is charged in a vessel of a reactor and allowed to stand. In the 
vapor diffusion method, a biopolymer solution and an insolubilizing agent 
solution are charged in two vessels separately and then diffused from one 
solution to the other solution. In the free interface diffusion method, a 
biopolymer solution and an insolubilizing agent solution are charged in a 
vessel of a reactor as separate layers forming the interface therebetween 
and ingredients of the solutions then diffused into each of the solutions. 
The batch-wise method is carried out by keeping an oversaturated biopolymer 
solution stationary to nucleate, crystallize, and grow biopolymer crystals 
from the solution. The oversaturated biopolymer solution containing a high 
salt concentration is prepared by the process of dissolving a biopolymer 
in water or an aqueous buffer solution, followed by adding a salt solution 
to the biopolymer solution. 
The vapor diffusion method is carried out by diffusing H.sub.2 O vapor (or 
an organic solvent vapor) in a biopolymer solution passing through the 
vapor phase equilibrated between the biopolymer solution and an aqueous 
salt solution (or an organic solvent solution) separated from each other 
in a closed system. When using an aqueous salt solution, the biopolymer 
solution is concentrated by diffusing water vapor from the solution to the 
aqueous salt solution because of a diluted concentration of the aqueous 
salt solution. When using an organic solvent solution, organic solvent 
vapor is dissolved into a biopolymer solution. In either case, the 
biopolymer solution becomes oversaturated by vapor diffusion. 
The free interface diffusion method is carried out by forming separate 
layers of a biopolymer solution and an aqueous salt solution or an organic 
solvent solution, one on top of the other. Crystallization and growth of 
the biopolymer crystals occur near the free interface of the solutions 
where oversaturation is attained, by diffusion with each other. 
As the buffer, acetic acid, phosphoric acid, trisulfuric acid buffers may 
be exemplified. As the neutric salt, ammonium sulfate, phosphate, sodium 
chloride, magnesium sulfate, cerium chloride, etc., may be exemplified. 
First, an example of the vapor diffusion method is described with reference 
to FIG. 6A. 
A 2% aqueous solution of sperm whale myoglobin (produced by Sigma Chemical 
Co., USA) was used as the biopolymer solution 24 and a saturated aqueous 
solution of ammonium sulfate as an insolubilizing solution 21. 
The pipes used in this operation are shown by thickened lines in FIG. 6A. 
The pump 30 was operated at a speed of 1 ml/min, and the valve 26 
appropriately switched such that the sampling times, by the valve 26, of 
the ammonium sulfate solution 21, the buffer 23 and the myoglobin solution 
24 are in the ratio of 70:5:25 and the resultant mixture is stirred by the 
stirrer 28 to obtain a 0.5% myoglobin solution. 
After connecting the valve 34 to the waste reservoir 35 to wash the pipe, 
the valve 34 was connected to the reactor 51 and 1 ml of the above 
solution supplied therein. At the same time, by operating the pump 29 at a 
speed of 1 ml/min, the saturated solution of ammonium sulfate 21 was sent 
through the valve 31 to the valve 32. 1 ml of the solution 21 was supplied 
to the reactor 51 after washing the pipe. As a result, the two solutions 
were charged in the two vessels A and B of the reactor 51, as shown in 
FIGS. 5 and 6A. During the above operation, excess gas in the reactor 51 
was discharged through the valves 33 and 31, as a result of the 
introduction of the solutions into the reactor 51. 
Subsequently, reaction solutions could be supplied to the other reactors 52 
to 61 respectively by switching the valve 32 to 34 in a similar manner to 
that described above. Here, if desired, different crystallization methods 
or conditions may be selected for any of the other reactors 52 to 61 by 
changing the operation of the pumps and the valves. Thus, different 
crystallization methods and/or conditions can be established in the same 
apparatus. 
The operations for the batch-wise method and the free interface diffusion 
method are shown in FIGS. 6B and 6C, respectively. 
Finally, the pipes were washed with the wash solution 22 and the waste 
removed and sent to the waste reservoir 35. 
The temperature in the reactors 51 to 61 was then controlled by the 
temperature control system 4 for a time period required for crystal 
growth. The growth of crystals in the reactors 51 to 61 was observed and 
recorded by the image recording system 7. The growth of crystals can be 
visually inspected, if necessary. 
The conditions for crystal growth in the three methods in the above 
experiments are shown in Table 1. In Table 1, the concentration of the 
myoglobin solution means that concentration in the reactor, and the 80% 
saturation of the ammonium sulfate solution means a mixture of the 100% 
saturated ammonium sulfate solution and the buffer in a ratio of 4:1. 
TABLE 1 
______________________________________ 
Concentration of 
Concentration of 
myoglobin ammonium sulfate 
Method solution solution 
______________________________________ 
Batch-wise method 
0.5 wt % 80% saturation 
Vapor diffusion 
0.5 wt % 80% saturation 
method 
Free interface 
0.5 wt % 100% saturation 
diffusion method 
______________________________________ 
A myoglobin single crystal obtained as a result of the above operation is 
shown as a photograph in FIG. 7. The size of this crystal is about 100 
.mu.m. 
According to the present invention, the operation of preparing biopolymer 
crystals is conducted automatically in accordance with a procedure 
preliminarily set in a central control system. As a result, the operator 
can be completely or almost freed from operation work. The crystal growth 
in a method according to the present invention is reproducible and 
further, different methods and/or conditions of preparing crystals can be 
conducted at the same time in the reactor system. 
By changing the temperature control, crystal growth at a gradually 
increased or decreased temperature can be carried out in the above 
apparatus, with a similar procedure. 
Another apparatus for use under zero gravity is described with reference to 
FIGS. 8A, 8B, and 9. The apparatus is similar to the apparatus in FIG. 2 
except for the reactor. FIGS. 8A and 8B are plane and sectional views of a 
reactor system and an image recording system of this embodiment. 
In FIGS. 8A and 8B, parts or components having similar functions to those 
of FIGS. 4A and 4B are denoted by the same reference numerals. In this 
apparatus, liquid supply pipes 80 (80a, 80b and 80c) penetrate the 
reactors 50 (51 to 61), each of which is a simple closed box and does not 
have two vessels as does a reactor in FIG. 3. Under zero gravity, if 
certain amount of liquid is introduced into a reactor 50 through a pipe 
80, the liquid forms a sphere outside the end of the pipe and the sphere 
is suspended from the pipe. Therefore, by introducing biopolymer and 
insolubilizing agent solutions through two neighboring pipes, e.g., 80b 
and 80c respectively, two spheres of the biopolymer and insolubilizing 
agent solutions are formed and these two spheres come into contact with 
each other, thus forming a free interface between the two solutions. FIG. 
8B shows this method. If a mixture of biopolymer and insolubilizing agent 
solutions is introduced through a pipe, the stationary standing batch 
method of preparation of crystals can be conducted. If biopolymer and 
insolubilizing agent solutions are introduced through two pipes 80a and 
80c which are separated from each other, two spheres of the solutions can 
be formed and spaced apart, enabling the vapor diffusion method of 
preparation of crystals to be carried out. 
FIG. 9 shows a perspective view of an apparatus for preparing biopolymer 
crystals, intended to be used in a free flyer which is placed in space by 
a space shuttle and recovered after experiments have been carried out. In 
FIG. 9, 101 denotes a supply unit, 102 a reactor unit, 103 an electric 
source and temperature control unit, 104 a recording and controlling unit, 
and 105 a data processing unit. The data processing unit 105 is connected 
to a communicating system of the free flyer. By providing the data 
processing unit 105, the apparatus for preparing crystals can be 
controlled on the earth in a desired procedure and can transmit images of 
crystal growth in the reactor to the earth, by communication between the 
free flyer and the earth.