U.S. patent application number 09/131729 was filed with the patent office on 2001-07-05 for dynamically controlled crystal growth system.
This patent application is currently assigned to UNIVERSITY OF ALABAMA. Invention is credited to BRAY, TERRY L., DELUCAS, LAWRENCE J., HARRINGTON, MICHAEL, KIM, LARRY J..
Application Number | 20010006807 09/131729 |
Document ID | / |
Family ID | 26672811 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006807 |
Kind Code |
A1 |
BRAY, TERRY L. ; et
al. |
July 5, 2001 |
DYNAMICALLY CONTROLLED CRYSTAL GROWTH SYSTEM
Abstract
Crystal growth can be initiated and controlled by dynamically
controlled vapor diffusion or temperature change. In one aspect,
the present invention uses a precisely controlled vapor diffusion
approach to monitor and control protein crystal growth. The system
utilizes a humidity sensor and various interfaces under computer
control to effect virtually any evaporation rate from a number of
different growth solutions simultaneously by means of an
evaporative gas flow. A static laser light scattering sensor can be
used to detect aggregation events and trigger a change in the
evaporation rate for a growth solution. A control/follower
configuration can be used to actively monitor one chamber and
accurately control replicate chambers relative to the control
chamber. In a second aspect, the invention exploits the varying
solubility of proteins versus temperature to control the growth of
protein crystals. This system contains miniature thermoelectric
devices under microcomputer control that change temperature as
needed to grow crystals of a given protein. Complex temperature
ramps are possible using this approach. A static laser light
scattering probe also can be used in this system as a non-invasive
probe for detection of aggregation events. The automated dynamic
control system provides systematic and predictable responses with
regard to crystal size. These systems can be used for microgravity
crystallization projects, for example in a space shuttle, and for
crystallization work under terrestial conditions. The present
invention is particularly useful for macromolecular
crystallization, e.g. for proteins, polypeptides, nucleic acids,
viruses and virus particles.
Inventors: |
BRAY, TERRY L.; (HOOVER,
AL) ; KIM, LARRY J.; (BIRMINGHAM, AL) ;
HARRINGTON, MICHAEL; (BIRMINGHAM, AL) ; DELUCAS,
LAWRENCE J.; (BIRMINGHAM, AL) |
Correspondence
Address: |
MERCHANT & GOULD P.C.
ATTN: DOUGLAS P. MUELLER
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
UNIVERSITY OF ALABAMA
BIRMINGHAM
AL
|
Family ID: |
26672811 |
Appl. No.: |
09/131729 |
Filed: |
August 10, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09131729 |
Aug 10, 1998 |
|
|
|
08719481 |
Sep 25, 1996 |
|
|
|
Current U.S.
Class: |
435/239 ;
435/235.1; 435/283.1 |
Current CPC
Class: |
C07K 2299/00 20130101;
Y10S 117/901 20130101; C12N 9/2462 20130101; Y10T 117/1008
20150115; C30B 29/58 20130101; C30B 7/00 20130101 |
Class at
Publication: |
435/239 ;
435/235.1; 435/283.1 |
International
Class: |
C12N 007/02; C12N
001/00; C12M 001/00 |
Goverment Interests
[0001] The work reflected in this application was supported by NASA
contract NAS8-40189.
Claims
What is claimed is:
1. An apparatus for crystal growth, comprising: a growth chamber
for containing a solution comprising a substance to be crystallized
and a solvent for the substance to be crystallized, whereby
evaporation of the solvent from the solution causes crystallization
of the substance to be crystallized; and a gas flow source in
communication with the growth chamber, for providing a gas flow to
remove evaporated solvent vapor from the growth chamber.
2. An apparatus according to claim 1, further comprising a gas flow
source control for changing the gas flow rate to the growth
chamber.
3. An apparatus according to claim 2, further comprising an
aggregation or nucleation detector for determining the onset of
aggregation or nucleation in the solution and creating a signal for
the gas flow source control to change the gas flow rate in response
to the onset of aggregation or nucleation.
4. An apparatus according to claim 1, wherein the reaction chamber
comprises first and second sections, wherein the solution is
located in the first section and the gas flow is provided to the
second section.
5. An apparatus according to claim 4, further comprising a barrier
between the first and second sections for preventing any
substantial impingement of the gas flow on the solution in the
first section.
6. An apparatus according to claim 5, wherein the barrier is a
plate with a central opening.
7. An apparatus according to claim 5, wherein the barrier is a
membrane that is permeable to the solvent vapor.
8. An apparatus according to claim 1, wherein the gas is
nitrogen.
9. An apparatus according to claim 1, further comprising a detector
for solvent vapor removed from the growth chamber.
10. An apparatus according to claim 1, wherein the solvent is water
and the apparatus comprises a sensor for measuring humidity of gas
removed from the growth chamber.
11. An apparatus according to claim 1, wherein the substance to be
crystallized is selected from the group consisting of a protein, a
polypeptide, a nucleic acid, a virus and a virus fragment.
12. method of growing crystals, comprising: dissolving a substance
to be crystallized in a solvent to form a solution; disposing the
solution in a growth chamber whereby solvent can evaporate from the
solution; and supplying a gas flow to the growth chamber to remove
evaporated solvent from the gorwth chamber.
13. The method of claim 12, further comprising analyzing the
solution to detect an onset of nucleation and changing the gas flow
rate upon detection of the onset of nucleation.
14. The method of claim 12, wherein the growth chamber comprises
first and second sections, with the solution being disposed in the
first section and the gas flow being provided to the second
section.
15. The method of claim 14, wherein direct impingement of the gas
flow on the solution is substantially prevented.
16. The method of claim 12, wherein the substance to be
crystallized is selected from the group consisting of a protein, a
polypeptide, a nucleic acid, a virus and a virus fragment.
17. The method of claim 16, wherein the substance to be
crystallized is a protein.
18. The method of claim 16, wherein the substance to be
crystallized is a polypeptide.
19. The method of claim 16, wherein the substance to be
crystallized is a nucleic acid.
20. The method of claim 16, wherein the substance to be
crystallized is a virus.
21. The method of claim 16, wherein the substance to be
crystallized is a virus fragment.
22. An apparatus for crystal growth, comprising: a growth chamber
comprising a container for containing a solution comprising a
substance to be crystallized and a solvent for the substance to be
crystallized, whereby a change in the temperature of the solution
causes crystallization of the substance to be crystallized; and a
control for changing the temperature of the solution.
23. An apparatus according to claim 22, further comprising a
nucleation detector for determining the onset of nucleation in the
solution and creating a signal for the control to change the rate
of temperature change in response to the onset of nucleation.
24. An apparatus according to claim 23, wherein the control
decreases the temperature of the solution prior to nucleation and
increases the temperature of the solution after nucleation.
25. An apparatus according to claim 23, wherein the control
increases the temperature of the solution prior to nucleation and
decreases the temperature of the solution after nucleation.
26. An apparatus according to claim 22, wherein the container is
capable of holding a solution volume of at least 50
microliters.
27. A method of growing crystals, comprising: dissolving a
substance to be crystallized in a solvent to form a solution;
disposing the solution in a container in a growth chamber; and
changing the temperature of the solution to cause crystallization
of the substance to be crystallized.
28. The method of claim 27, further comprising analyzing the
solution to detect an onset of nucleation and changing the rate of
temperature change upon detection of the onset of nucleation.
29. The method of claim 28, wherein the temperature is decreased
prior to nucleation and increased after nucleation.
30. The method of claim 28, wherein the temperature is increased
prior to nucleation and decreased after nucleation.
31. The method of claim 27, wherein the substance to be
crystallized is selected from the group consisting of a protein, a
polypeptide and a virus fragment.
32. The method of claim 31, wherein the substance to be
crystallized is a protein.
33. The method of claim 31, wherein the substance to be
crystallized is a polypeptide.
34. The method of claim 31, wherein the substance to be
crystallized is a nucleic acid.
35. The method of claim 31, wherein the substance to be
crystallized is a virus.
36. The method of claim 31, wherein the substance to be
crystallized is a virus fragment.
37. The method of claim 29, wherein the temperature is held at a
constant level after the period of increase.
38. The method of claim 30, wherein the temperature is held at a
constant level after the period of decrease.
Description
BACKGROUND OF THE INVENTION
[0002] Protein structural information has proven beneficial for
understanding structure/function relationships and for applications
such as structure-based drug design. X-ray crystallography is the
predominant technique used to obtain three-dimensional protein
structure information. A critical component of this technique is
the growth of high quality, well ordered crystals of the target
protein. Advances in x-ray diffraction equipment, data collection
methods, and computational capabilities have progressed to the
point where the growth of high quality crystals is often the rate
limiting step for the determination of three-dimensional protein
structures. Many different techniques have been used in the attempt
to grow high quality protein crystals. The most widely used protein
crystal growth technique, vapor diffusion, utilizes a growth
solution containing protein and a precipitating agent. A popular
vapor diffusion configuration, typically described as the
hanging-drop or Linbro method (MCPHERSON, JR., A. (1982),
Preparation and Analysis of Protein Crystals (Wiley, New York)-see
FIG. 1), uses a reservoir solution containing precipitant and a
buffered protein/precipitant solution that "hangs" from a sealed
cover slip positioned over the reservoir. The initial solution
conditions are such that water vapor diffuses from the protein
solution into the reservoir solution, thereby increasing the
concentration of the protein beyond its solubility point. One
significant limitation of the traditional vapor diffusion technique
is that the evaporation of water from the growth solution (within a
particular geometry) is fixed by the starting concentrations of the
solution components (see FIG. 2). Thus, the rate at which the
approach to supersaturation of the growth solution occurs is fixed,
even if modification of this evaporation rate is desirable, and
this technique suffers from the inability to control the vapor
equilibration process once the experiment is initiated.
[0003] The vapor diffusion technique has been used successfully to
grow protein crystals in the microgravity environment of NASA's
Space Shuttle, with space flight hardware called the Vapor
Diffusion Apparatus or VDA, (HERRMAINN, F. T., and HERREN, B. J.
(1990) "Crystal Growth Apparatus", U.S. Pat. No. 4,919,899 and
SNYDER, R. S.; HERREN, B. J.; CARTER, D. C.; YOST, V. H.; BUGG, C.
E.; DELUCAS, L. J.; SUDDATH, F. L. (1991) "Macromolecular Growing
Sytems", U.S. Pat. No. 5,013,531). The original Vapor Diffusion
Apparatus (VDA) was used to grow protein crystals in the
microgravity environment of NASA's Space Shuttle. However, its
concept and design have proven not to be optimal and it has
specific limitations. For example, during crystal growth and
nucleation, the vapor diffusion profile is fixed by the starting
solution concentrations. Also no modification of the experiment is
possible and photography during crystal growth is sporadic and
non-isothermal. Although significant, the results with the Vapor
Diffusion Apparatus are not optimal as evidenced by the following
statistics. Only 25% of all proteins flown in the VDA produced
crystals that diffracted better than any crystals grown on Earth.
40% of protein flown produced crystals which did not diffract
better and 35% produced no crystals. Clearly there is need for
methods and devices to improve the success ratio. Also,
investigations have been underway (SMITH, H. W. & DELUCAS, L.
J. (1991), J. Crystal Growth 110 137; WILSON, L. J. & SUDDATH,
F. L. (1992), J. Crystal Growth 116 414) in the attempt to produce
systems that will allow control over the evaporation profile of a
growth solution. Early experiments showed that simply slowing down
the evaporation rate of a growth solution generally produces a
smaller population of larger crystals than can be obtained with
traditional vapor diffusion techniques. Recent results from a large
number of experiments shows this effect to be consistent not only
for lysozyme, but for other proteins as well.
[0004] While vapor diffusion has been (and still is) a very popular
technique, it has not always proven to be the best method for a
given protein, and hence a wide range of other approaches have been
used as a means to obtain high quality protein crystals. Another
protein crystal growth technique, temperature, has only begun to be
extensively explored in recent years. This technique utilizes the
variable solubility versus temperature that some proteins exhibit
for a given solution condition as a means for initiating and
controlling crystal growth. Though this approach offers promise
when compared to other techniques for controlling the rate of
growth to produce high quality protein crystals, this method is not
without limitations. Several proteins have been crystallized
successfully using temperature (BAKER, E. N. and DODSON, G. (1970),
J. Mol. Biol. 54, 605; SHOTTON, D. M. , HARTLEY, B. S., CAMERMAN,
N. and HOFMAN, T. (1968), J. Mol. Biol. 32, 155; HANSON, A. W.,
APPLEBURY, M. L., COLEMAN, J. E. and WYCKOFF, H. (1970), J. Biol.
Chem. 245, 4975; MCPHERSON, JR., A. and RICH, A. (1972), Biochem.
Biophys. Acta 285, 493), and recent developments in custom
instrumentation and devices that screen protein solubility versus
temperature improve the usefulness of temperature as a strategic
method for growing protein crystals (CACIOPPO, E., MUNSON, S. and
PUSEY, M. L. (1991), J. Crystal Growth 110, 66).
[0005] Despite this, the approach to finding suitable conditions
that yield high quality protein crystals predominantly has been a
trial and error process, where more than one thousand
crystallization conditions are typically screened, often without
success. Several systems have been constructed to aid the growth of
protein crystals. These systems vary in complexity from simple
hand-held devices (EISELE, J. -L. (1993), J. Appl. Cryst. 26, 92)
to complex robotic systems that simply prepare and monitor
different conditions (Cox, M. J. and WEBER, P. C. (1988), J.
Crystal Growth 90, 318; CHAYEN, N. E., STEWART, P. D. S., MAEDER,
D. L. and BLOW, D. M. (1990), J. Appl. Cryst. 23, 297). Only a few
systems have attempted to achieve control over the dynamics of
protein crystal growth (WILSON, L. J., BRAY, T. L. and SUDDATH, F.
L. (1991), J. Crystal Growth 110, 142; CASEY, G. A. and WILSON, W.
W. (1992), J. Crystal Growth 122, 95) by altering the rate at which
water is removed from the growth solution. Other investigations
have been underway (SMITH, H. W. and DELUCAS, L. J. (1991), J.
Crystal Growth 110, 137; WILSON, L. J. and SUDDATH, F. L. (1992),
J. Crystal Growth 116, 414) in the attempt to produce systems that
will allow control over the evaporation profile of a growth
solution. Early experiments showed that simply slowing down the
evaporation rate of a growth solution generally produces a smaller
population of larger crystals than can be obtained with traditional
vapor diffusion techniques. However, they have not offered true
dynamic control of the protein crystal growth process.
SUMMARY OF THE INVENTION
[0006] The present invention provides a system for dynamic control
of crystal growth, particularly for difficult-to-crystallize
macromolecular substances such as proteins, polypeptides, nucleic
acids, viruses and virus fragments. The nucleic acids include DNA,
RNA and fragments of DNA and RNA. While reference is made hereafter
to protein crystal growth, it should be understood that these
teachnigs will be equally applicable to other macromolecular
substances. Dynamic control of protein crystal growth (DC/PCG) has
operational advantages that include the ability to separate protein
crystal aggregation and/or nucleation from the post nucleation
protein crystal growth phase and the potential for limiting the
number of nucleation sites. Supersaturation conditions necessary
for the aggregation and /or nucleation of proteins are often
significantly greater than those needed for subsequent growth of
the crystal. In order to minimize problems during the subsequent
growth phase caused by the higher supersaturation necessary for
nucleation, it is desirable to separately control the nucleation
and growth environments. With DC/PCG, one has the capability to
vary post-nucleation growth kinetics. One also has the capability
to optimize crystal growth conditions in subsequent experiments.
With respect to microgravity experiments, DC/PCG also minimizes
protein sample quantities and minimizes astronaut crew time in
experiment operation.
[0007] The present invention provides systems with increased
capacity and versatility for the growth of protein crystals by
vapor diffusion or temperature. Temperature adjustments are
advantageous in providing a non-invasive means of controlling the
supersaturation environment of protein nuclei. Additionally, laser
light scattering data from the growth medium allows the aggregation
state of the protein to be evaluated non-invasively and provides
for dynamic control of the crystallization process. These systems
achieve truly dynamically controlled protein crystal growth system
that can be used for both terrestrial and microgravity
experiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 illustrates a Linbro box hanging drop vapor diffusion
chamber of the prior art.
[0009] FIG. 2 is a graph showing drop volume versus time for a
typical vapor diffusion experiment in a Linbro box.
[0010] FIG. 3 is a block diagram of an example for dynamic control
protein crystal growth (DC/PCG) via vapor diffusion with
evaporation rate controlled by nitrogen gas flow.
[0011] FIG. 4 is a schematic illustration of a dynamically
controlled vapor diffusion system.
[0012] FIG. 5(a) and (b) show a thermal conductivity humidity
detector and its circuit.
[0013] FIG. 6 is an enlarged illustration of one of the elements
shown in FIG. 4.
[0014] FIG. 7 is a flowchart for software for controlling the vapor
diffusion system.
[0015] FIG. 8 is a sectional side view of a crystal growth chamber
used in the system of FIGS. 4 and 6.
[0016] FIGS. 9(a) and (b) show lysozyme crystal growth results at
varying evaporation rates.
[0017] FIGS. 10(a) and (b) show thaumatin crystal growth results at
varying evaporation rates.
[0018] FIG. 11 illustrates an example of a dynamically controlled
vapor diffusion control/follower system.
[0019] FIG. 12 illustrates a further example of a crystal growth
chamber.
[0020] FIG. 13 shows evaporation profiles overlaying typical static
laser light scattering signal as aggregation occurs.
[0021] FIG. 14 shows results for lysozyme crystal growth at
different evaporation rates, under conditions where the evaporation
was terminated upon detection of aggregation and/or nucleation
(triggered) and where evaporation continued after the onset of
nucleation (non-triggered).
[0022] FIG. 15 is a block diagram of a system for dynamic control
protein crystal growth (DC/PCG) via temperature induction.
[0023] FIG. 16 is an enlarged view of a growth chamber from the
system of FIG. 14.
[0024] FIG. 17 is a scematic illustration of a dynamically
controlled temperature system.
[0025] FIG. 18 is a schematic illustration of laser light
scattering with nucleation chamber.
[0026] FIG. 19 is circuit diagram for a temperature controller that
can be used with the controlled temperature system.
[0027] FIG. 20 is a flowchart for software for controlling the
controlled temperature system.
[0028] FIGS. 21(a)-(d) show examples of computer generated
temperature profiles useful with the temperature induction protein
crystal growth systems.
[0029] FIGS. 22(a)-(d) show plots of voltage and solution
temperature versus experiment time for lysozyme crystals.
[0030] FIGS. 23(a)-(d) are micrographs showing lysozyme crystals
obtained from experiments whose results are graphed in FIGS. 22(a)
- (d).
[0031] FIG. 24(a) and (b) show plots of voltage and solution
temperature versus .experiment time for bovine insulin aggregates
and/or nuclei.
[0032] FIG. 24(c) and (d) show plots of voltage and solution
temperature versus experiment time for porcine insulin aggregates
and/or nuclei.
[0033] FIG. 25 shows temperature crystallization data for the
protein neurophysin.
[0034] FIG. 26 illustrates a dynamically controlled temperature
control/follower system.
[0035] FIG. 27 shows details of the control/follower system.
[0036] FIGS. 28(a) and (b) show results from crystallization of
lysozyme with a modified version of the temperature crystal growth
method.
[0037] FIG. 29 is a diagram of an example of an interface between
the Space Shuttle (STS) and a vapor diffusion dynamically
controlled protein crystal growth system.
[0038] FIG. 30 is a diagram of an example of an interface between
the Space Shuttle (STS) and a temperature induction dynamically
controlled protein crystal growth system.
DETAILED DESCRIPTION
[0039] The disclosure of U.S. Ser. No. 08/432,914 filed May 1, 1995
is incorporated herein by reference in its entirety.
[0040] The present invention is directed to a method and apparatus
for the dynamic control of the crystal growth process. In
particular, the present invention is directed to crystal growth
systems based on vapor diffusion or temperature changes for
initiating crystallization from a solution. While the present
invention is useful for crystallization of inorganic and organic
substances in general, the present invention is particularly useful
for crystallization of macromolecular substances such as proteins,
polypeptides, nucleic acids (for example DNA, RNA and fragment of
DNA and RNA), viruses and virus fragments, especially proteins. The
present invention is useful in crystallization studies for
structure-based drug design targets and for determining structures
of substances to be inhibited in treating diseases. In addition,
the present invention is useful for work with complexes of various
macromolecular substances, for example with inhibitors, and such
related substances should be considered to included in the
definition of the macromolecular substances.
[0041] Ordinarily, for macromolecular substances the substance to
be crystallized will be dissolved in water, but the present
invention is equally useful for other solvents as needed. The
dynamic control of the present invention can be based on feedback
obtained by observation of the solution being crystallized. Such
feedback can be obtained by using monitoring techniques such as
laser light scattering, real time video , turbidity measurements,
interferometry, diffraction, ultrasonography, scintillation,
polarization, schlieren optics, ellipsometry, holography and raman
spectroscopy. It will be noted that some of these monitoring
systems, e.g. laser light scattering, are not capable of
distinguishing between aggregation and nucleation in the solution.
This is not critical in the context of the present invention, and
the detection of either event is suitable. The present invention
can be used advantageously for microgravity crystallization work,
for example in the Space Shuttle, as well as for terrestial
work.
Vapor Diffusion Based Systems
[0042] The present invention can provide dynamic control for
crystallization in the traditional "hanging drop" type
configuration as well as in a "container" configuration. In each
case, the solvent evaporation rate can be controlled carefully to
respond to the progression of the crystallization. The solvent
evaporation rate can be controlled by means of a variable gas flow.
It is particularly desirable to avoid having the gas flow impinge
directly upon the crystallization solution. One way to accomplish
this is to have the crystallization reaction chamber separated into
two sections, a solvent evaporation section and a purging section,
separated by a barrier that substantially prevents direct
impingement of the gas flow from the purging section on the
crystallization solution but permits passage of solvent vapor. The
rate of solvent evaporation from the crystallization solution can
be controlled by varying the gas flow through the purging
section.
[0043] The removal of solvent from the crystal growth solution can
be checked by monitoring the gas removed from the purging section.
In the case of an aqueous solvent, this can be done by monitoring
the relative humidity of the purged gas. It is possible to
multiplex a number of reaction chambers to one humidity sensor,
allowing a large number of experiments to be performed
simultaneously. It also is possible to handle a number of reaction
chambers using a "control/follower" concept, where one chamber (the
control) is monitored actively and other chambers (the followers)
are acted upon in response to events occurring in the control
chamber. A static laser light scattering system is one example of a
useful a sensor for detecting aggregation events occurring in
growth solutions to trigger a response, such as changing the
evaporation rate of one or more of the crystal growth solutions.
Laser light scattering is an important diagnostic tool to monitor
protein crystal nucleation. It constitutes a sensitive,
nonintrusive and real-time diagnostic for dynamic control of
protein crystal growth. It can be adapted for most protein crystal
growth configurations and can provide predictive information
important to protein crystal growth.
[0044] A first example of a dynamically controlled vapor diffusion
system (DCVDS) is based on the "hanging drop" vapor diffusion
methods that have yielded relatively high success rates for
obtaining protein crystals. This system incorporates a controlled
flow of gas, dry nitrogen (N.sub.2) gas as one example, instead of
a reservoir solution to extract water, via the vapor phase, from
the growth solution. This allows precise control over the rate at
which the growth solution "equilibrates." In the traditional
"hanging drop" vapor diffusion experiment, water diffuses rapidly
during the early stages of the experiment and subsequently slows
down asymptotically as the experiment progresses. Depending upon
the solution components, complete equilibration can occur over a
range of 3 to 30 days (Fowlis et al., (1988), J. Crystal Growth 90,
117). The present system described allows the equilibration rate to
be varied at virtually any rate. While nitrogen gas is useful,
other gases can be used as desired, and examples include
non-reactive gases such as He, Ne, Xe, Ar and Kr. In addition,
other gases such as CO.sub.2 can be used if they do not have an
adverse impact on the solution being crystallized, or if measures
are taken to reduce any adverse impact such as buffering against pH
changes.
[0045] FIG. 3 is a block diagram of a suitable system for protein
crystal growth effected by vapor diffusion with nitrogen gas to
control the evaporation rate. The sample chamber can be
instrumented with laser light scattering (LLS), video monitoring
and a humidity detector to moderate nitrogen flow. The growth
occurs under constant temperature conditions via isothermal
temperature control. The system may be monitored and controlled by
computer.
[0046] Referring now to FIG. 4, the system screens up to 40
different evaporation profiles simultaneously, using growth
chambers in which the growth solution is deployed as a hanging
drop. Gas flow in and out of each chamber is controlled by a pair
of valves (available for example from Lee Company of West Brook
Connecticut). The amount of water evaporated from each growth
solution at any given time is measured quantitatively using a
microvolume thermal conductivity detector (TCD) (available for
example from Gow-Mac of Greenbelt Maryland). FIGS. 5 (a) and (b)
show a thermal conductivity detector (TCD) used for humidity
monitoring and its circuitry.
[0047] The chambers may be grouped in sets of 10 with each set
multiplexed to one TCD. The ten chambers fit onto one baseplate
(see FIG. 6), which provides structural support for the chambers,
the valves for each chamber, the multiplexing manifolds, and an
electrical umbilical. The growth chambers may be controlled and
monitored by a microcomputer, for example a Macintosh Quadra 950
microcomputer using custom software written in the program language
LabView to monitor each set of 10 growth chambers, allowing for
individual or replicate profiles to be executed in any given
chamber. Data acquisition and interface boards are used for all
analog and digital input/output functions. FIG. 7 provides a
flowchart for a software system useful for carrying out the
controlling and monitoring functions.
[0048] FIG. 8 shows the crystallization chamber. The
crystallization chamber is designed to simplify the set up and
recovery of experiments and can be made of any suitable material,
such as glass, metal or polymeric materials such as acrylic
polymers and polysulfone. Materials such as acrylics that are
substantially optically transparent are preferred. The growth
solution is deployed as a droplet 40 on a standard glass coverslip
42 and placed on the chamber body 46. A threaded cap 44 fits over
the coverslip, securing it to the chamber body. Two O-rings 48 (e.g
{fraction (1/16)}inch in cross section) provide an airtight seal
for the chamber when the cap is screwed down finger-tight. The
chamber body has two quick-disconnect valves for connection to the
purge gas supply and removal lines 50 and 52. This allows removal
from the baseplate (e.g., for photography) while maintaining
integrity of the airtight seal.
[0049] The chamber has dual compartments or sections to aid in
controlling evaporation from the drop surface. The first section 54
contains the hanging drop of crystal growth solution and can be
considered as the crystallization reaction section. The second
section 56 is in communication with the gas supply and removal
lines, and solvent evaporating from the growth solution is removed
by means of the gas supplied to and removed from the second section
56. The sections 54 and 56 are separated by a barrier 58 that acts
to prevent any substantial direct impingement of the gas on the
growth solution. However, the barrier permits the passage of
evaporated solvent from the first section to the second section.
The barrier can be in the form of a washer whose central opening
permits vapor to pass to the second section while its body prevents
any substantial flow of gas from the second section to the first
section. In this case, the washer can be made of metal, plastic or
any other suitable material. The barrier can also take the form of
a fabric or membrane made of a material or otherwise formed to be
permeable to solvent vapor. In this case, the barrier can extend
completely between the two sections, without the need for a central
opening. When water is the solvent, a polymeric material such as
"GORETEX" that permits the passage of water vapor can be used for
the fabric or membrane. Other useful materials include a nylon mesh
screen material, filter paper or other porous cellulose material,
and nucleopore filters. In the case of filter paper, it is
preferred that the material be one that does not absorb the
evaporated solvent. In the case of the nucleopore filter, the pore
size should be sufficiently large to permit passage of the vapor
phase molecules to pass therethrough. If the gas flow is permitted
to impinge directly on the growth solution, the solvent vapor
gradient may become too steep, resulting in the formation of a
large number of nucleation sites (a "shower") at the solution
surface. This is undesirable since it tends to produce large
numbers of small crystals.
[0050] A microvolume thermal conductivity detector (TCD) is used to
quantitate the amount and rate of water evaporated from each growth
solution. The difference in thermal conductivities between dry
N.sub.2 gas and moist N.sub.2 gas produces a signal that can be
quantitatively related to the amount of moisture in the purge
volume. The integration of this signal with respect to time for
sequential purges of a given chamber allows the total water
evaporated from each chamber to be determined and provides feedback
to the host microcomputer so that virtually any desired evaporation
profile may be followed.
[0051] A custom software program (e.g. as shown in FIG. 7) written
in the graphical programming language LabVIEW (available from
National Instruments of Austin, Tex.) running on a Macintosh Quadra
950 (Apple Computer) microcomputer controls all aspects of the
system. A user interface allows various experimental control
parameters to be entered prior to execution. The program controls
the purges of each chamber based upon the evaporation profile
entered before execution using feedback from the TCD. This allows
virtually any evaporation profile to be followed. The program also
performs all data acquisition functions, digital output functions,
and writes the cumulative data file to the internal hard disk for
subsequent analysis.
EXAMPLE 1
[0052] Hen egg white lysozyme was obtained from Calbiochem. NaCl,
NaOAc, and glacial acetic acid were obtained from Fisher
Scientific. A 50 mM acetate buffer was prepared by dissolving NaOAc
in 18 Mohm deionized water. The pH was adjusted to 4.7 using
glacial acetic acid. Lysozyme was dissolved in buffer and then
dialyzed several times against fresh buffer to remove any salt from
the source protein. This solution was then concentrated using
Amicon microconcentrators to create a stock solution at 40 mg/mL. A
7% (w/v) stock solution of NaCl was prepared by dissolving the NaCl
salt in buffer. All solutions were filtered through 0.22 micron
Whatman filters.
[0053] Thaumatin, NaK tartrate, and ADA
(N-[2-Acetamido]-2-iminodiacetic acid) were obtained from Sigma.
Glacial acetic acid was obtained from Fisher Scientific. A 68 mg/mL
stock Thaumatin solution was prepared by dissolving the protein in
18 Mohm deionized water. An ADA buffer was prepared by dissolving
ADA salt in 18 Mohm deionized water and adjusting the pH to 6.5
with glacial acetic acid. A 0.75 M stock solution of NaK tartrate
was prepared by dissolving the NaK tartrate salt in ADA buffer
solution. All solutions were filtered through 0.22 micron Whatinan
filters.
[0054] For each protein, lysozyme and thaumatin, the growth
solution was deployed onto silanized glass coverslips by mixing 10
microliters of each of the stock protein and crystallizing agent
solutions (20 microliters total). Each coverslip was then placed on
a chamber and sealed with a screw-down cap. Linear evaporation
profiles were performed to determine the effects of evaporation
rate on crystal growth results. Solutions were evaporated at 0.041,
0.083, 0.2, 0.34, 0.45, and 1.25 microliters/hr to half the
original volume.
[0055] Results from a large number of experiments for the two
proteins show that clear, systematic trends are observed as a
function of evaporation rate (see FIGS. 9 (a) and (b) and 10(a) and
(b)). As the evaporation rate is increased, larger populations of
smaller crystals are observed. Since the supersaturation level
required for nucleation to occur is higher than that needed to
sustain crystal growth, the observed results are likely related to
the length of time that the solution resides in the nucleation
region. For a given evaporation rate, once nucleation occurs,
crystal growth can proceed for those nuclei formed. This crystal
growth will begin depleting protein from the solution. However, if
the supersaturation level is increasing faster due to evaporation
than it is decreasing due to crystal growth, then nucleation will
continue to occur until crystal growth depletes the protein
concentration below the supersaturation level required for
nucleation. Hence, faster evaporation rates should maintain a high
supersaturation level longer than slower evaporation rates, leading
to increased crystal populations. Also, larger crystal populations
should generally yield smaller crystals since the amount of protein
in the growth solution is fixed and cannot sustain crystal growth
to large sizes for a large number of crystals.
[0056] Additionally, comparative x-ray analysis of crystals grown
using this hardware system with crystals grown by traditional
methods have been undertaken. Results from the vapor
diffusion/nitrogen systems indicate that longer evaporation
profiles generally produce larger crystals and that the larger
crystals generally produce enhanced x-ray diffraction. Also,
crystals of equal size grown at different evaporation rates show a
wide and overlapping variation of diffraction characteristics
(signal to noise, resolution).
[0057] In a second aspect, the present invention can utilize a
control/follower system to monitor and control a number of
dynamically controlled vapor diffusion crystallization chambers
(the follower chambers) by means of the information collected for a
single chamber (the control chamber). A dynamic controlled vapor
diffusion control/follower system (DCVDC/FS) (see FIG. 11) has been
constructed that provides dynamic control of the supersaturation
level during the crystal growth process. This device uses concepts
similar to the previously described system that controls
supersaturation levels. The DCVDC/FS system also incorporates a
laser light scattering (LLS) subsystem as a noninvasive probe for
detecting aggregation events occurring in a growth solution. The
detection sensor provides feedback to the controlling microcomputer
so that the evaporation profile can be modified in real time in an
attempt to optimize crystal growth. The illustrated system has six
growth chambers arranged in a control/follower configuration with
the control chamber 60 containing the LLS system which is connected
to the humidity sensor (e.g. a TCD). Some or all of the remaining
chambers are evaporated at a rate that mimics that of the control
chamber.
[0058] Each of the six growth chambers contain the following
components: cuvette, upper glass purge chamber, three piece
aluminum housing, and thumbscrew. The control chamber (see FIG. 12)
also contains two fiber optic cables, one for the 5 mW, 633 nm
He--Ne laser (available from Melles Griot of Irvine, Calif.) and
photodetector (available from Hammamatsu of Bridgewater, N.J.). In
the aluminum housing, the top component is milled out to accomodate
the upper chamber cell and gas flow lines. Additionally, a threaded
portal is provided through which a thumbscrew any be deployed
against the upper chamber cell to sealit against the cuvette. A
cylindrical portal may be milled through the thumbscrew to provide
access for visual observation. The middle component of the aluminum
housing is milled through to provide a pathway for the cuvette mid
upperchamber to meet when sealed against each other. This component
essentially acts as a removable spacer to allow the cuvette to be
removed easily from the lower component. The lower component is
milled to accept the cuvette. It also has portals milled out for
two fiber optic cables. A portal through the bottom of the lower
component is provided to allow for visual observation. Typically, a
light source will provide backlight through the bottom of the lower
component.
[0059] The growth chamber is a commercially available glass
fluorimeter cuvette. The portion of the cell where the actual
growth solution is contained has a cross sectional dimension of 2
mm.times.10 mm and is 12.5 mm high. A second chamber is sealed on
the top of the cuvette to aid in controlling the evaporation of
water from the growth solution. The two chambers are spearated by a
barrier that is permeable to solvent vapor and substantially
prevents any direct impingement of the purge gas on the growth
solution as in the previous embodiment. Transmission and detection
of the laser light is accomplished via optical fibers. The entire
assembly resides in a three component aluminum housing which
provides mechanical support for the upper chamber cell and the
laser and photo detector fiber optic cables. The aluminum housing
also provides a mechanism for sealing the upper chamber to the
growth cell (via a thumb screw).
EXAMPLE 2
[0060] Hen egg white Lysozyme was obtained from Calbiochem. NaCl,
NaOAc, and glacial acetic acid were obtained from Fisher
Scientific. A 50 mM acetate buffer was prepared by dissolving NaOAc
in 18 Mohm deionized water. The pH was adjusted to 4.7 using
glacial acetic acid. Lysozyme was dissolved in buffer and then
dialyzed several times against fresh buffer to remove any salt from
the source protein. This solution was then concentrated using
Amicon microconcentrators to create a stock solution at 45 mg/mL. A
7% (w/v) stock solution of NaCl was prepared by dissolving the salt
in buffer. Equal volumes of the stock protein and NaCl solutions
were mixed and passed through a filter loop to produce a solution
clean enough to use for laser light scattering. This loop consisted
of a peristaltic pump, tubing, and a 0.22 micron Whatman filter.
The solution was filtered for 30 minutes and then deployed directly
into the cuvettes.
[0061] 200 microliters of the growth solution is deployed in the
cuvette, inserted into the chamber assembly and sealed. Initial
experiments with this device have revolved around the use of linear
evaporation profiles in the early stages of the experiment. Once
nucleation is detected by the light scattering system, the profile
is modified for half of the chambers, with the original profile
continued for the remainder. This allows a direct comparison
between the results obtained from continued evaporation at the
original linear rate versus modification of that rate in response
to the light scattering sensor. Two different evaporation rates in
the early stages of the experiments were investigated. The growth
solutions were evaporated at 0.83 and 1.389 microliters/hr.,
respectively, until the laser light scattering sensor was triggered
at the detection of nucleation. At this point, half of the chambers
ceased evaporation while half continued at the original evaporation
rate (to half the original volume). There is a distinct change in
the slope of the photodetector response as aggregation occurs. This
change is subsequently responded to by the computer system that
controls the evaporation profile. Typical experimental profiles and
a typical response of the photodetector to aggregation are shown in
FIG. 13. There is a distinct change in the slope of the
photodetector response as aggregation occurs. This change is
subsequently responded to by the computer system that controls the
evaporation profile.
[0062] Preliminary results for this system indicate that
modification of the evaporation profile in response to light
scattering detection of nucleation yields improved crystallization
results. Generally, modification of the evaporation profile after
nucleation detection, in this case a reduction in evaporation rate,
results in a smaller population of slightly larger crystals. A
faster initial rate of evaporation also affects the crystal growth
results, yielding a larger population of smaller crystals than
observed at the slower initial evaporation rate (FIG. 14). As
explained earlier, it is likely that growth solutions in which
nucleation is detected and evaporation ceased have fewer nuclei,
allowing the remaining protein in solution to "feed" growth of
these crystals to a larger size. The growth solutions that continue
evaporating likely reside in a nucleation region longer, producing
more crystals which cannot grow as large.
Temperature Based Systems
[0063] The present invention also can be used as a dynamic control
system that controls the supersaturation state of materials to be
crystallized from solution via precise temperature changes.
Temperature can be precisely controlled and used as a tool to
control the population and size of the crystals being obtained,
e.g. protein crystals. It also is possible to use a non-invasive
sensor to detect aggregation events and modify the temperature
program for a given experiment in response to the sensed events.
This aspect of the present invention also is useful for the
control/follower concept. This again is accomplished by monitoring
aggregation events in the control chamber and controlling the
temperature of the follower chambers in response to the behavior of
the experiment in the control chamber. In general, the temperature
of the growth solution will be lowered or raised (depending upon
whether the substance in question shows decreased solubility with
lower temperature or higher temperature) until nucleation is
initiated. At this point, the temperature change can be stopped or
reversed to inhibit further nucleation that could hinder the growth
of larger crystals. In some cases, it will be desirable to reverse
the temperature change and then hold the solution at a constant
temperature when it reaches a desirable temperature for crystal
growth.
[0064] FIG. 15 is a block diagram of an example of a system for
protein crystal growth effected by temperature change. This diagram
outlines the major components for dynamically controlled
temperature system (DCTS). The sample chamber in the center circle
is instrumented with a thermal electric device (TED) for
temperature change and control and with laser light scattering via
laser probe and detector probe. The system is monitored and
controlled via computer. In this example, the growth chamber has
two compartments, with a laser probe being centered in one of the
chambers. FIG. 16 is an expanded view of the growth system depicted
in FIG. 15.
[0065] As shown in FIG. 17, this system uses precise temperature
adjustments to initiate, monitor, and control the growth of protein
crystals. Temperature is manipulated via miniature thermoelectric
devices (TEDs) under computer control to change the temperature in
virtually any desired manner. Detection of aggregation events is
accomplished using a static laser light scattering system (LLS),
which acts as a sensor to provide feedback to the controlling
microcomputer. All aspects of the systems are monitored and
adjusted by a 486 microcomputer containing a data acquisition
interface card. Custom software written in QuickBASIC allows the
user to set a particular temperature program for a given
experiment.
[0066] An example of the DCTS apparatus is shown schematically in
FIG. 18. The copper jacketed nucleation chamber houses a 10 mm
glass cuvette (Starna Cells, Inc., Atascadero, Calif.) which
contains 200 microliters of the appropriate growth medium.
Generally, a volume of at least 50 micorliters is needed since it
can be difficult to couple the laser into a smaller volume. The use
of a container for the growth solution permits better thermal
contact and resultant improvement in temperature control for the
solution. The copper-jacketed nucleation chamber is surrounded by
Teflon encased in an aluminum block. The nucleation chamber, in
conjunction with a Marlow 5000 series temperature controller
(available from Marlow Industries, Dallas, Tex.), an IBM compatible
PC, thermoelectric device, and type T thermocouple, allow precise
temperature control of the protein growth medium. The static laser
light scattering geometry is also shown. Laser light from a
helium-neon 633 nm, 5 mW Melles Griot laser is directed into the
protein solution via a fiber optic cable. A second fiber optic
cable is placed at 90.degree.to the incident laser fiber and
collects scattered light that is carried to a photodetector. The
output of the photodetector is returned to the controlling CPU in
the form of an analog signal. The controlling CPU compares voltages
and modifies temperatures as defined by the experimental
parameters. The nominal error for temperature control associated
with this experimental set up is +/- 0.1.degree.C. with a maximum
error of +/-0.25.degree. C. A circuit diagram of a particularly
useful temperature control system is shown in FIG. 19.
[0067] The growth solution is deployed into a cuvette, which is
then placed in the copper jacket and sealed with a cover.
Temperature is controlled using a small TED powered by a commercial
TED controller/power supply (Marlow Industries) under the direction
of a microcomputer. Custom software allows user defined temperature
programs to be executed and modified in response to the LLS signal.
A flowchart for the software system is shown in FIG. 20.
[0068] A custom software program (e.g. as shown in FIG. 20) written
in QuickBasic running on a 486 microcomputer is used to control all
aspects of the system. For a particular experiment, this program
allows the user to enter the prenucleation temperature profile, the
photodetector signal level which designates that aggregation events
are occurring, and the postnucleation temperature profile. The
programs can control the temperature profile for the reaction in
any desired manner. For example, one can allow very simple
temperature profiles to be executed (SIMPRO) while the another
allows much more complex temperature profiles to be executed
(COMPRO) in an attempt to optimize crystal growth. Examples of
thermal profiles that can be used for temperature induction of
protein crystal growth are shown in FIG. 21. These profiles are
generated by the thermal control computer software developed in
house. Simple straight forward profiles (Simpro) and complex
profiles (Compro) can be designed with the software. The
software/microcomputer perform all control and data acquisition
functions, recording the data files to an internal hard disk.
[0069] The temperature based system can be applied to any material
to be crystallized. It is believed to be particularly useful for
macromolecular materials such as proteins, nucleic acids and virus
particles, especially proteins. In addition, it should be
recognized that the proteins that will be most suitable for
temperature controlled crystallization are those that show a
.DELTA.B.sub.22/.DELTA.T value of at least 2.times.10.sup.5 mol
ml/g.sup.2 deg, where B is the osmotic second virial coefficient, a
measure of protein-protein specific interactions in a specific
solution condition. Examples include lysozyme, hen eg albumin,
pepsin, alpha-chymotrysinogen A, equine serum albumin, bovine serum
albumin, thaumatin, neurophysin-dipeptide complex, bovine insulin
(T3R3), edestin, porcine insulin (T3R3) and human
insulin-4-hydroxybenzamide.
EXAMPLE 3
[0070] Chicken egg white lysozyme was obtained from Calbiochem.
Bovine insulin (pancreas), and porcine insulin (pancreas) were
obtained from Sigma. All proteins were used as received without
further purification. Zinc acetate dihydrate was obtained from
Sigma Chemical Company. Acetone, trisodium citrate sodium acetate
trihydrate, and sodium chloride (all certified, ACS reagent grade)
were obtained from Fisher Scientific. Lysozyme was crystallized
using solubility data collected by Cacioppo and Pusey. The
crystallizing medium consisted of 25 mg/mL of lysozyme and 2.5%
sodium chloride in 50 mM sodium acetate buffer at pH=4.4. Both
bovine and porcine insulins were crystallized from 0.05 M trisodium
citrate and 0.0075 M zinc acetate in the presence of 0.75 M sodium
chloride, 15% acetone, and 5% water at pH=6.4 (M. M. Harding, et
al. (1966) J. Mol. Biol. 16, 30:323-327). All growth media were
centrifuged and filtered prior to use.
[0071] Temperature-induced bulk crystallization was accomplished by
incubating the crystallizing medium at an initial temperature at
which the protein is in a quasi-equilibrium state. At this
temperature, aggregate size distribution is stable and stable
baseline voltages are obtained. After steady state data were
accumulated, temperature was decreased at programmed rates to
increase the level of supersaturation and affect protein
aggregation. The aggregation event was defined as a percentage
above steady state voltage. After aggregation was observed, the
temperature was increased at programmed rates to a final growth
temperature.
[0072] The present system successfully used static laser light
scattering to detect the aggregation of lysozyme and effectively to
decouple the nucleation and growth phases of lysozyme crystal
growth. This was accomplished by inducing nucleation at low
temperature/high supersaturation conditions, observing an increase
in the total intensity of the scattered light as measured by SLS,
and increasing the temperature to dissociate some of the aggregates
and provide for a better environment for the growth of remaining
aggregates. The change in scattering voltage with respect to
temperature changes is shown in the double Y graphs of FIG. 22.
These graphs depict detector voltage and temperature versus
experiment time. These representative plots all show that
scattering voltage (particle size) increases with decreasing
temperature (protein aggregation) and that the onset of voltage
increase is directly correlated with the decrease in temperature.
As the temperature is increased, particle size decreases
(aggregates dissociate) and the decrease in the magnitude of the
scattering voltage is correlated with the onset of the temperature
increase. Crystal populations and morphologies obtained from the
experiments described in FIG. 22 are shown in the micrographs of
FIG. 23.
[0073] These photomicrographs clearly demonstrate that aggregation
at a low temperature/high supersaturation condition followed by an
increase in temperature to an environment of lower protein
supersaturation affects the population of microscopic crystals
produced. Approaches to both the nucleation temperature (the
temperature where aggregation first occurs) and the final growth
temperature affect both the number of microcrystals produced at the
nucleation temperature and the number of microcrystals that are
dissociated when the temperature is increased. Porcine and bovine
insulins were also crystallized by DC/PCGT. Static light scattering
data demonstrates that insulin aggregates rapidly with decreasing
temperature and that the particle size distribution could be
reversed with an increase in temperature. This is shown in FIG.
24(a)-(d). Small populations of insulin crystals ranging in size
from 0.2 to 0.4 mm were produced. Details of the experimental
conditions are presented below.
[0074] With reference to FIG. 24(a), Bovine insulin (Sigma, 10 mg)
was dissolved in 1.5 mL of 0.02 M HCl. The following reagents were
added in the order listed: 150 microliters of 0.15 M zinc acetate,
750 microliters of 0.2 M trisodium citrate, 510 microliters of
acetone, 90 microliters of water, and 0.18 g of sodium chloride.
The pH was increased to about 11 with microliter volumes of dilute
sodium hydroxide to solubilize all medium components and then was
backtitrated to 6.4 with microliter amounts of dilute hydrochloric
acid. The medium was heated at 40.degree. C. for several minutes
and then centrifuged at room temperature for one hour. The growth
medium was incubated at 22.degree. C. in the nucleation chambers
and then the temperature was decreased to 10.degree. C. at a rate
of 0.5.degree. C./min. After nucleation was detected (1.15.times.
baseline voltage), the temperature was increased to the final
growth temperature of 28.degree. C. at a rate of 0.5.degree.
C./min. A small population of crystals measuring 0.2 mm.times.0.2
mm was obtained after four days of incubation at the final growth
temperature.
[0075] With reference to FIG. 24(b), bovine insulin (Sigma, 8 mg)
was dissolved in 1.5 mL of 0.02 M HCl. The following reagnets were
added in the order liseted: 150 microliters of 0.15 M zinc acetate,
750 microliters of 0.2 M trisodium citrate, 510 micorliters of
acetone, 90 microliters of water, and 0.18 g of sodium chloride.
The pH was adjusted to 6.4 and the solution was heated for a few
minutes at 40.degree.C. to ensure that all medium components were
in solution. The growth medium was centrifuged for one hour at
40.degree.C., filtered over an Anotop filter and then incubated at
40.degree.C. in the nucleation chambers for one hour. Then the
temperature was decreased from 40.degree.C. to 10.degree.C. at a
rate of 0.02.degree.C./min. After nucleation was detected
(1.15.times. baseline voltage), the temperature was increased to
the final growth temperature of 28.degree.C. at a rate of
0.5.degree.C./min. This resulted in a large population of 0.1
mm.times.0.1 mm crystals by day 6 of incubation at the final growth
temperature
[0076] With reference to FIG. 24(c), porcine insulin (Sigma, 10 mg)
was dissolved in 1.5 mL of 0.02 M HCl. The following reagents were
added in the order listed: 150 microliters of 0.15 M zinc acetate,
750 microliters of 0.2 M trisodium citrate, 510 microliters of
acetone, 90 microliters of water, and 0.18 g of sodium chloride.
The pH was increased to .about.9 with microliter amounts of dilute
sodium hydroxide to solubilize all components and then the solution
was backtitrated to pH=6.4 with dilute hydrochloric acid. The
solution was centrifuged for one hour at 40.degree. C., filtered
over an Anotop filter, incubated at 22.degree. C. in the nucleation
chamber for one hour and then the temperature was decreased from
40.degree. C. to 10.degree. C. at a rate of 0.5.degree. C./min.
After nucleation was detected (this was set at 1.15.times. baseline
voltage that was accumulated during the one hour incubation at
40.degree. C.), the temperature was increased to the final growth
temperature of 28.degree. C. at a rate of 0.5.degree. C./min. A
small population of 0.25 mm.times.0.20 mm crystals were obtained
after one day at the final growth temperature.
[0077] With reference to FIG. 24(d), porcine insulin (Sigma, 10 mg)
was dissolved in 1.5 mL of 0.02 M HCl. The following reagents were
added in the order listed: 150 microliters of 0.15 M zinc acetate,
750 microliters of 0.2 M trisodium citrate, 510 microliters of
acetone, 90 microliters of water, and 0.18 g of solid sodium
chloride. The pH was increased by the addition of microliter
amounts of dilte sodim hydroxide to solubilize all medium
components and then backtitrated with microliter amounts of dilute
hydrochloric acid to pH=6.4. The mediuk was centrifuged at room
temperature, filtered over an Anotop filter and incubated at
30.degree.C. for one hour in the nucleation chambers. The
temperature was decreased to the nucleation temperature of
10.degree.C. at a rate of 0.05.degree.C./min. After nucleation was
detected (1.15.times. baseline voltage), the temperature was
increased to the final growth temperature of 28.degree.C. at a rate
of 0.25.degree.C./min. A small population of crystals measuring 0.3
mm.times.0.3 mm was obtained after five days of incubation at the
final growth temperature.
[0078] FIG. 25 presents data collected from the temperature
induction system of Example 3 on the protein, neurophysin. As the
ramp rate decreased, or in other words, as the temperature change
occurred over a longer period of time, the crystal size
increased.
[0079] Thus, static laser light scattering is one example of a
system that can be used to detect the aggregation of lysozyme and
insulin, and the particle size response of these proteins to
changes in temperature is rapid. This allows a decoupling of the
nucleation and growth stages of crystal growth. In addition, these
results demonstrate that temperature can have a profound effect on
the population and size of crystals that are obtained for a given
experiment. The rate at which the temperature is changed as well as
the target nucleation temperature both can affect the size and
population of crystals obtained for a given protein. This effect
has been examined for several different proteins including
lysozyme, insulin and insulin complexes (from different species),
and neurophysin. Results from the temperature induction studies and
systems indicate it is possible to demonstrate that crystal
nucleation can be detected by laser light scattering in microliter
volume samples and can be reversed via temperature change (.sigma.
decreased, where .sigma. is the ratio of protein concentration over
protein solubility). The results show that nucleation temperature
varies in a qualitative way that is predictable and reproducible as
a function of protein solution variables. They also demonstrate
that it is possible to investigate various post-nucleation growth
profiles by dynamically controlling temperature to change .sigma..
In the temperature induction studies, we found that the change in
the baseline scattering definition that triggers a change in
temperature profile(s) varied from 15% to 45% of the light
scattering background. In these studies, T.sub.nuc definition is
the target nucleation temperature. It was found that large
aggregate/nuclei were more difficult to dissociate and that lower
T.sub.nuc definition resulted in fewer, larger crystals.
[0080] FIGS. 26 and 27 show a temperature based dynamic control
system incorporating the basic technical features described above,
but does differ in some of the specific hardware. Temperature is
controlled with a programmable water bath while the growth chamber
may be a 200 microliter polysulfone cylinder. It includes a
control/follower arrangement to investigate the reproducibility of
crystals grown by temperature in which the status of one chamber is
used to determine the actions taken on other chambers. The control
chamber incorporates the static laser light scattering system for
detecting aggregation events, while the three follower chambers do
not. The crystallization chambers are housed in an insulated
container, which circulates water from a programmable water bath to
control the temperature. The previously described custom software
(SIMPRO or COMPRO) is used to drive the experiments.
[0081] A thermocouple provides temperature feedback to the host
microcomputer. The incident laser beam enters the control growth
chamber from the bottom of the growth chamber through a fiber optic
cable. Scattered light is collected at 90.degree.to the incident
beam via a second fiber optic cable that is carried to a
photodetector. Foam insulation surrounds the assembly to maintain a
given temperature during a reaction.
EXAMPLE 4
[0082] Hen egg white lysozyme was obtained from Calbiochem. NaCl,
NaOAc, and glacial acetic acid were obtained from Fisher
Scientific. A 50 mM acetate buffer was prepared by dissolving NaOAc
in 18 Mohm deionized water. The pH was adjusted to 4.7 using
glacial acetic acid. Lysozyme was dissolved in buffer and then
dialyzed several times against fresh buffer to remove any salt from
the source protein. This solution was then concentrated using
Amicon microconcentrators to create a stock solution at 120 mg/mL.
A 4% (w/v) stock solution of NaCl was prepared by dissolving the
salt in buffer. Equal volumes of the stock protein and NaCl
solutions were mixed and centrifuged for one hour. The supernatant
was then filtered through a 0.2 micron Anotop filter.
[0083] 160 microliters of the growth solution is deployed into each
growth cell, and then the cells are inserted into the chamber
assembly and sealed with caps. From the initial temperature T1
(22.degree.C. in this example) the temperature is ramped at some
rate R1 (0.5.degree. C./minute down in this example) towards the
target nucleation temperature T2 (3.degree.C. in this example). If
nucleation is detected by the LLS system, then the temperature ramp
is modified before reaching the target temperature. Upon detecting
nucleation or reaching the target nucleation temperature, the
temperature is then ramped at some rate R2 (0.5.degree. C./minute
up in this example) to a final growth temperature T3 (13.degree.C.
in this example). Other more complex temperature profiles are
possible as defined by the experimenter.
[0084] Initial experiments with this system were performed with two
proteins, lysozyme and thaumatin. The results obtained using this
system demonstrate good reproducibility between cells run under
identical conditions (FIG. 28). Both the number and size of
crystals obtained are similar in all four cells. This demonstrates
that a control/follower arrangement can be used with success to
grow crystals of comparable size and quantity in several different
cells while actively monitoring (with static laser light
scattering, LLS) only one of the identically controlled cells. This
approach eliminates the need to use multiple sensors to control
duplicate (identical) experiments.
[0085] It can be seen that the present invention can utilize two
methods, vapor diffusion and temperature, to control the nucleation
and growth of protein crystals. Either method can be used
successfully to gain control over the crystal growth process in a
manner previously unavailable.
[0086] The vapor diffusion approach has shown that varying the
evaporation rate of the growth solutions systematically affects the
size and number of the crystals obtained. Faster evaporation rates
generally lead to larger populations of smaller crystals than
slower evaporation rates. Inclusion of a non-invasive sensor (LLS)
has been used to detect aggregation, at which point the initial
evaporation rate is modified, resulting in a smaller population of
larger crystals than obtained without dynamic response. These
results are likely related to the length of time that a given
growth solution resides at a supersaturation level where nucleation
occurs.
[0087] The temperature based protein crystal growth systems follow
an approach where the crystal growth solution is maintained in a
container, rather than as a hanging drop, and use precise control
of temperature via TED's or a water bath to induce nucleation and
control crystal growth. A static laser light scattering system is
used as a diagnostic to detect aggregation and the temperature
modified to optimize crystal growth. A control/follower
configuration has been used whereby information obtained by
monitoring one chamber can be used to effect similar results in the
remaining chambers.
[0088] While the present invention is useful for terrestial crystal
growth, development of these systems for microgravity studies
should improve the success of protein crystal growth on the space
shuttle and/or the international Space Station. The flight
experiment would be via a dynamically controlled protein crystal
growth technique which uses either of the two methods, vapor
equilibration or temperature, to control and optimize the
crystallization process. This would be accomplished by controlling
supersaturation prior to and after the nucleation event, which can
be detected via laser light scattering. Video information can also
be used to evaluate crystal data so that subsequent experiments
could be altered in an attempt to optimize results. FIGS. 29 and 30
are respectively diagrams of examples of the system interfaces for
the vapor diffusion systems (DC/PCG-V) and the temperature systems
(DC/PCG-T) with the Space Shuttle (STS). The systems used for space
shuttle work should be made with components approved under the
relevant NASA guidelines. In FIG. 26, the indication STES stands
for Single Thermal Enclosure System, which also is known as
CRIM.
[0089] While a detailed description has been provided above, the
present invention is not limited thereto, but rather is defined by
the following claims.
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