Patent Application
20100144011
This invention relates to a method for growing crystals under microgravity conditions, and more particularly to grow crystals such as protein crystals under microgravity conditions. Moreover, the present invention relates to a method of producing a single crystal. More particularly, the present invention relates to a method of producing a large single crystal having a low density of crystalline defects.
Protein crystal growth under microgravity conditions results in substantially increased crystal size and quality. The application of the microgravity environment is the subject of several ongoing investigations which aim to increase the size and internal order of protein crystals. Numerous successful applications of the microgravity environment to the growth of high quality protein crystals have been well documented. This is extremely important, since the ability to produce high quality protein crystals has been the limiting step in a number of important macromolecule structural problems. Crystallization of proteins is an important requirement in determining protein structure. Protein crystallography is used to ascertain the three-dimensional molecular structure of protein crystals. This is essential for understanding the biological functions attributed to these macromolecules. The physical shape and folding of a protein is of increasing importance to drug companies interested in rational drug design. Drug molecules are designed to fit exactly into a binding site of a macromolecule, thus blocking its function in a given disease pathway. Producing higher quality crystals results in more accurate modelling of the 3-dimensional protein structures and consequently more efficacious drugs. This accuracy is referred to as the resolution of the structure. The larger and more perfect crystals provide the highest resolution.
Presently, the field of macromolecular crystallography is undergoing a major technological revolution which permits more efficient and difficult structure determinations. These improvements coupled with the advances in recombinant technologies are providing an increase in the number of structures determined yearly. Typically, the growth of large, high quality protein crystals using ground-based methods requires numerous crystallization surveys to identify and maximize the proper growth conditions.
With regard to making a crystal (preferably a single crystal structure), various methods have been conventionally known. For example, a melt growth method in which a melt is slowly solidified, and a solution growth method in which a solution of raw materials is cooled gradually have been provided. To avoid crystalline defects or in-homogeneity in composition and make a perfect single crystal, a zero-gravity environment such as in space or orbit has been utilized. For instance, a melt or solution is cooled under a floating state without using any container, and a crystal is made. This method is called a container-less method, and it is a stable method for making a highly pure crystal because any contamination from a container can be completely avoided. A large crystal is also made because a melt of large size can be supported without any container under the zero-gravity environment. At the beginning of crystal growth experiments in space, a perfect crystal without any defect or in-homogeneity was expected to be realized on the grounds that convection due to gravity does not occur and crystal growth proceeds in a melt or solution without any influence of disturbance.
One important field of such a space experiment is the growth of protein crystals under the microgravity environment. The growth of protein crystals is an important as well as fundamental step for determining the molecular structure and for investigating the relationship between the structure and function of the protein molecules. Based upon the determination of the molecular structure, it is expected to design the proteins having a desired function. This is one of the major goals of protein engineering.
In view of the foregoing problems, the experiments in space for growing the protein crystals under microgravity environments attracts attention of various researchers, as such a microgravity environment does not cause the convection when growing the protein crystals. Perfect crystals are difficult to achieve on Earth. Ambient gravity and turbulence disrupt crystal formation in that terrestrial samples mix as a result of gravity-driven convective flow. Therefore a microgravity environment promotes better crystal formation, in part due to the lack of turbulence and mixing within a liquid or gaseous sample during crystal formation. Spacecraft in low Earth orbits can provide a microgravity environment that is convection- and sedimentation-free for the study and applications of fluid-based systems. With the advent of the Space Shuttle, scientists had regular access to such environments and many experiments were initiated, including those in protein crystallization. After many trials it became clear that for several proteins, crystallization in a microgravity environment resulted in bigger and better quality crystals. The generation of perfect crystals can sometimes be the limiting factor in determining a protein's structure. By eliminating variables such as gravity, crystals are able to form slower and more precise in space. Temperature can be a significant variable in biological macromolecule and small molecule crystallization. Temperature often influences nucleation and crystal growth by manipulating the solubility and supersaturation of the sample. Thus the control of temperature during crystal production is essential for successful and reproducible crystal growth of proteins with temperature dependent solubility. An advantage is that a temperature gradient provides precise, quick, and reversible control of relative supersaturation. Using temperature in addition to standard crystallization variables (such as sample concentration, reagent composition and concentration, and pH), can increase the probability of producing crystals as well as uncover new crystallization conditions for a sample. Protein solution temperature can be used to carefully manipulate crystal nucleation and growth. This control can also be used to etch or partially dissolve then grow back the crystal in an attempt to improve crystal size, morphology, and quality. Temperature control is noninvasive and can manipulate sample solubility and crystallization with altering reagent formulation.
The present embodiment relates to a method of producing a 3D protein crystal structure from a purified protein, wherein the protein is isolated from a purified Geobacillus sp.strain T1. The purified protein includes thermostable T1 lipase and thermostable F16L lipase, whereby the thermostable T1 lipase and thermostable F16L lipase is obtained at a temperature of between 16° C. and 70° C. Preferably, it is understood that the T1 lipase has a working protein concentration of at least 2 mg/ml and the F16L lipase having a working protein concentration of at least 4 mg/ml.
Accordingly, it is also understood that the present invention relates to a method of a) growing 3D protein crystal structure in a gravity environment; and growing 3D protein crystal structure in a microgravity environment. This present method relates to the preparation of a protein solution; a reservoir solution; and as a result of the method a protein droplet is obtained. The protein droplet obtained for the T1 and F16L lipase shows a working protein insert of between 5 μl and 40 μl, whereby the insert includes a protein concentration of at least 1.25 μl and a reservoir solution of at least 1.25 μl. (Preferably the protein concentration is between 1.25 μl and 10 μl; and the reservoir solution between 1.25 μl and 10 μl). Moreover, the protein solution includes T1 lipase or F16L lipase, a buffer solution having a working pH of at least pH 8.5 and glycerol between 3% and 5%.
Yet another embodiment of the present invention relates to a 3D crystal structure (preferably a 3D thermostable lipase T1 and/or a thermostable F16L lipase structure) having the means to grow in the gravity environment whereby the 3D structure having a working temperature of at least 20° C. and for at least 8 days. Following to this, the 3D protein crystal structure of thermostable T1 lipase and/or thermostable F16L lipase is obtained from Geobacillus sp.strain T1. It is understood that the T1 lipase having a size of at least 1.8 Å under the gravity environment and at least 1.35 Å under microgravity environment, where else, the F16L lipase having a size of at least 1.8 Å under gravity environment and at least 1.7 Å under microgravity environment.
a), 5(b), 5(c) and 5(d) represent size of T1 lipase in space.
a,
6(b), 6(c) and 6(d) represent size of F16L lipase in space and ground control.
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
In a microgravity environment the absence of convective heat transfer produces some undesirable results. Solution cooling and the resulting supersaturation proceed by diffusion. Solution adjacent to a cooled wall, or fluid-gas interface will have a localized increase in its supersaturation. The proximity of solid surfaces or fluid-gas interfaces to the nucleated crystals tends to distort the mass diffusion fields around the crystals. This results in the nucleation of uncontrolled numbers of crystals on the surfaces, such as the walls of the container, or on the free surface of the liquid. The nucleation of large numbers of crystals at one time limits the size to which the crystals will grow by rapidly depleting the solution of solute. This is particularly troublesome in protein crystal growth where the production of large crystals is the purpose for using microgravity. Protein crystal growth under microgravity conditions results in substantially increased crystal size and quality. The application of the microgravity environment is the subject of several ongoing investigations which aim to increase the size and internal order of protein crystals.
The object of the present invention is to develop a suitable protein crystallization process on earth and on zero-gravity. Moreover, the invention relates to preferred conditions to develop protein crystals or 3D crystal structures. Furthermore, the preferred embodiment of the present invention relates to an evaluation of 3D crystal growth under microgravity (zero-gravity) and on earth. In addition, the invention also relates to the quality of protein crystal/s grown on earth and zero-gravity. The present invention provides a new and improved method for growing protein crystals and for screening crystallization conditions in solution crystal growth. As such, the principal object of the present invention, which will be described subsequently in greater detail, is to provide a new and improved method for growing protein crystals and for screening crystallization conditions in solution crystal growth which has all the advantages of the prior art and none of the disadvantages.
It is still further an object of the present invention to provide a new and improved method for growing protein crystals and for screening crystallization conditions in solution crystal growth which can be used to screen for crystal growth conditions for any variety of small-molecule crystals. It is yet another object of the present invention to provide a new and improved method for growing protein crystals under microgravity conditions and for screening crystallization conditions in solution crystal growth which greatly reduces the total manpower required for conducting experiments with the present invention.
The followings are the preferred methods used in the preparation of 3D crystal structure on ground level (earth) and under zero-gravity (space/microgravity). At least two Thermostable lipase were used in the presences of the present invention. The two Thermostable lipase that was utilized is T1 lipase (hereafter “T1”) and F16L lipase (hereafter “F16L”). The T1 and F16L were optimized between 16° C. and 60° C. with an optimum working temperature of 20° C. The said Thermostable lipase was isolated from Geobacillus sp. Strain T1. The Geobacillus T1 (preferably known as Geobacillus T1 & Geobacillus zalihae T1) is understood to be having an accession number of EMBL/GenBank/DDBJ Accession No: A7367764 and NBRC 101842 and could be obtained from the Department of Biotechnology and Molecular Sciences, University Putra Malaysia. Furthermore, the T1 and F16L are having a protein concentration between 2.5 mg/ml and 4.0 mg/ml.
The following methods are segmented into pre-flight activity; in-flight activity and post-flight activity. The pre-flight activity includes the followings: preparation of purified protein/s, wherein the preferred protein/s is T1 and F16L lipase. This purification process is conducted at ground level. As a result, two purified protein/s were obtained at the end of this method (purified T1 lipase and F16F lipase). Accordingly, during the in-flight activity, preparation to insert the T1 and F16L lipase into a cell barrel was conducted. At this stage, observation in the size of the protein was noted. Furthermore, during the post flight activity, verification on the size of the protein was conducted in a further experiment. A comparison results between the size of the protein at ground level and micro-gravity was compared.
Pre-Flight Activities:—Preparation of Purified Protein (Ground).
a) Two proteins (T1 and F16L) which was successfully crystallized in the laboratory of the Department of Biotechnology and Molecular Sciences, University Putra Malaysia. An Expression method then later followed by purification was conducted. Pure protein with specified protein concentration was prepared. Herewith a purified T1 and F16L lipase is obtained.
b) Placing the purified protein/s into a sealed eppendorf tubes and keeping (precipitating solution/s) into Universal bottles (for precipitant solutions). The above were packed in a packing container under dry-ice shipment at 0° C. (0 to −20° C., dry ice condition).
c) Crystallization of T1 lipase was conducted, whereby protein samples will be centrifuge at 14,000 rpm to remove any precipitate. Using a suitable hardware, the protein was first inserted into the Cell Barrel through a top surface of a Cell Body by carefully pressing into place with a clean firm object or the ground handle. The Cell Barrel was rotated at 180° to a fill-in position in order to prepare sample loading. Pipette the precipitating solution (about 500 μl) into the PPT Reservoir. Dropping, Chromex into the precipitating solution and pressing until the surface of the Chromex is parallel with the top surface of the PPT Reservoir. To ensure supersaturation small amounts of Reservoir solution will be added until no more is absorbed and place PPT Reservoir back into the Assembly. Accesses Cap will be temporarily remove from the same experiment location. Desired amount of T1 lipase and desired amount of the precipitant solution (Table 2) will be pipette into the Protein Insert with 1:1 ratio. PI will gently mix the two solutions and place the Access Cap back into a closed position. All steps will be repeated until all chambers are filed. After all chambers are filled, PI will rotate the cell barrel 90° where the lever will be aligned with the red color mark. The experiment now will be at deactivated position and are prepared 24 hrs prior to launch which then being transferred to the engineering team for leak test and packaging preparation. At this stage and prior to launch the PCS kit must be kept at a temperature of 10 to 25° C.
d) Crystallization of F16L lipase was conducted. Protein samples will be centrifuge at 14,000 rpm to remove any precipitate. Using a suitable hardware, the protein was first inserted into the Cell Barrel through a top surface of a Cell Body by carefully pressing into place with a clean firm object or the ground handle. The Cell Barrel was rotated at 1800 to a fill-in position in order to prepare sample loading. Pipette the precipitating solution (about 500 μl) into the PPT Reservoir. Dropping, Chromex into the precipitating solution and pressing until the surface of the Chromex is parallel with the top surface of the PPT Reservoir. To ensure supersaturation small amounts of Reservoir solution will be added until no more is absorbed and place PPT Reservoir back into the Assembly. Accesses Cap will be temporarily remove from the same experiment location. Desired amount of F16L lipase and desired amount of the precipitant solution (Table 2) will be pipette into the Protein Insert with 1:1 ratio. PI (Principal Investigator) will gently mix the two solutions and place the Access Cap back into a closed position. All steps will be repeated until all chambers are filed. After all chambers are filled, PI will rotate the cell barrel 90° where the lever will be aligned with the red color mark. The experiment now will be at deactivated position and are prepared 24 hrs prior to launch which then being transferred to the engineering team for leak test and packaging preparation. At this stage and prior to launch the PCS kit must be kept at a temperature of 10 to 25° C.
In-Flight Activities:—In the Space Station
a) A space representative or preferably an astronaut will activate the crystal growth experiments by rotating the cell barrel at about 90° whereby the lever being rotated from one indication mark to another indication mark, preferably a red color mark pointing to green marker. During the activation, a protein chamber is rotated for the purpose of its vapour to have the means of contacting or sealing with the reservoir. Water molecules will migrate from protein droplet through vapour space into a concentrated reservoir. As the volume of the protein droplet decreases, the concentration of protein increases and protein crystals are formed. As the experiment continues, it relates to the crystals growing larger.
b) After a successful launch and activation process, the hardware should be kept at ambient temperature and stable position. It should not be disturbed except for the required daily observation and should be in correct orientation at all times.
c) Before the end of the mission, the astronaut will deactivate the experiment by rotating the chamber 90° back to its original position (i.e. align the lever from green marker to the red marker) to reseal the samples before return to Earth. The samples are remaining sealed until it is returned to laboratory for further analysis. It is important that the crystals should be unshaken and in a position that ensures survivability during landing and after landing until handed back to the PI.
d)
Post-Flight Activities:
a) At the landing site the PCS Kit is retrieved and the samples are transported to a suitable laboratory for further analysis, wherein the samples are carried under ambient temperature (10° C.-25° C.).
b) Ground based activities: A synchronous ground control activity will be conducted for the pre-flight and in-flight activities at the same time under similar conditions. Crystal formed will then be subjected to X-ray diffraction for structure determination.
c) The samples containing crystals (T1 and F16L lipase crystal/s) will be studied using X-ray diffraction. To facilitate successful removal of protein crystals from the cell growth assembly, the cell barrel should be fully in an activated position. PPT reservoir will be removed from the bottom of the cell and the top of growth cell assembly will be gently rest flat surface. The Protein Insert side of the ground tool will be slowly place into the assembly and firmly push until the protein insert is out of the assembly and resting on the clean surface. The protein insert should now be accessible to the open environment and using a small hair-loop or other protein crystal tool will be gently and carefully remove from the protein insert for analysis.
d) Crystallization holds the molecules still so that the researchers can aim a single-electron X-ray beam at them. The electrons refract off the electrons of a molecule and create a pattern of dots on an X-ray film or digital camera chip. The pattern can then be converted to a density map that is the same general shape as the molecule. The expected high quality crystals to be crystallized in space may contribute significantly to the increase in the diffraction data which are necessary to obtain higher spectral resolutions for better structural information. With this structural information, PI can determine how the protein functions and can design new enzymes that target specific function.
Equipment Description
The PCS experiment requires the following equipment:
A payload, provided by the PI, is composed of:
The kit is consisting of one (1) Protein Assembly (AAN-PCS-01), one (1) HOBO Temperature Data Recorder (AAN-PCS-02) and one (1) Nomex Bag (AAN-PCS-03).
a. One (1) Protein Assembly (AAN-PCS-01).
b. One (1) HOBO Temperature Data Recorder (AAN-PCS-02).
The HOBO® is an autonomous data (temperature) recorder, powered by small internal Manganese Dioxide Lithium battery (1x CR2032 each). These data recorders are routinely flown aboard the NSTS/ISS. The HOBO® data recorders were flown aboard STS-95, 93, 106, 100, 110, 112. The batteries are vacuum tested to <0.1 psi for >6 hours, inspected for leaks, and voltage is measured both unloaded and loaded prior to flight integration. Batteries will not be accessible on orbit and cannot be replaced by the crew. HOBO® data is stored in non-volatile memory and therefore, battery life does not constitute the ‘operational life’ of the payload. HOBO is stored inside a heat-welded ESD bag.
c. One (1) Nomex Bag (AAN-PCS-03).
It is used to store the Protein Assembly and HOBO during the transport to and from the ISS-RS. This is a bag which created from a type of fiber with an extraordinary combination of high-performance heat- and flame-resistant properties, as well as superior textile characteristics.
Table 2 and 3 represents properties of the T1 lipase and F16L lipase. Table 4 represents the comparison of 3D crystal structure obtained on earth and in space. Table 4 shows the comparison of T1 and F16L lipase in space and ground control (earth). Following to this,
Table 4 represents the comparison pf 3D crystal structure obtained on earth and in space
Table 5 represents data collected for T1 lipase in space
Table 6 represents data collected for F16L lipase in space
As described in detail above, the present invention permits the production of a high grade single crystal without any crystal defect or inhomogeneity in composition which has fewer slips or twins based on strains and less contamination with impurities. A large-sized single crystal can also be produced. A zero-gravity of microgravity environment such as in outer space can be utilized effectively.
| Number | Date | Country | Kind |
|---|---|---|---|
| PI 2008 4994 | Dec 2008 | MY | national |
