Patent Application
20090111711
The present invention relates in general to the field of biotechnology and, in particular, to a microplate and methods for simultaneously screening a plurality of protein crystallization solutions and producing diffraction quality protein crystals in a vapor-diffusion environment in a high-density high-throughput format.
Various publications, which may include patents, published applications, technical articles and scholarly articles, are cited throughout the specification in parentheses, and full citations of each may be found at the end of the specification. Each of these cited publications is incorporated by reference herein, in its entirety.
Innovative technologies and advancements in experimental techniques have enabled researchers to rapidly increase both the number of newly identified genes and the number of three-dimensional structures of biological macromolecules. There have been significant improvements in the sequential process of gene expression, protein purification, crystallization, and structure determination, but crystallization remains as one of the major bottlenecks in crystal structure determination. To address that issue, a number of different high-throughput protein-crystallization methods have been proposed and a number of automated crystallization systems have been developed (Stevens 2000; Sugahara and Miyano 2002; Sulzenbacher et al. 2002; Watanabe et al. 2002; Hosfield et al. 2003; Hui and Edwards 2003; Stojanoff 2004; Hiraki et al. 2006). For example, the Oryx 6 (Douglas Instruments, Ltd., Berkshire, UK) can set up 96-wells in 12 minutes for sitting-drop vapour diffusion and the Syrrx system can set up 2880 drops for vapour diffusion per hour (Hosfield et al. 2003; Hiraki et al. 2006).
When compared to microbatch and hanging drop methods, sitting-drop vapour-diffusion methods and microplates have advantages for high-throughput crystallization applications. Advantages include easy observation of crystallization drops, easy harvesting of crystals from the drops, and easy handling of the microplates with standard robotics and liquid handling devices (Hiraki et al. 2006). Numerous sitting drop microplates are commercially available at low cost from a number of different vendors, including Hampton Research, Greiner, and Corning. Others, such as Emerald Biostructures Inc., Structural Genomics Inc., and UAB Research Foundation have designed their own microplates or microarrays for custom applications (U.S. Pat. Nos. 6,039,804; 6,656,267; and 7,214,540). Some examples of sitting drop protein crystallography microplates or microarrays are briefly discussed below.
In U.S. Pat. No. 7,214,540, there is disclosed a method of screening protein crystal growth conditions with microchambers having a volume from about 0.001 nl to about 250 nl. Also disclosed is a method that employs a microarray with a plurality of wells or reservoirs as shown in
The microplate of the present invention has advantages over other available crystallography microplates. The microplate of the present invention is in a high-density 1536-well format with 768 functional wells, thus allowing for a truly high-density high-throughput screen using a sitting-drop vapor-diffusion method. The standard 1536-well format allows for facile robotic handling of the microplate and compatibility with a wide range of liquid handling systems. Furthermore, using wells of equal size with bottoms aligned in the same plane at the bottom of the wells allows for facile imaging with an inverted light microscope while at the same time allowing manipulation and harvesting of crystals from above. In a preferred embodiment, in which the bottoms of the wells are flat, microscopic images of the wells can be very rapidly screened because the bottoms of the wells are in a single focal plane. It should also be noted that the decreased reservoir to droplet ratio volumes of the high-density high-throughput format should lead to faster equilibration rates and more rapid protein nucleation and crystal growth compared to using other available crystallography microplates (Santarsiero et al. 2002).
The microplate and methods of the present invention also have an advantage over the microarray and methods described in U.S. Pat. No. 7,214,540. By using the microplate of the present invention with 8 μl maximum volumes it is possible to use protein solution volumes of about 1 μl or volumes as much as 2 μl, thus the method of the present invention allows for growth of diffraction quality crystals during a high-density high-throughput screen. The crystals obtained directly from the screen are suitable for analysis by x-ray, thus eliminating the need to reproduce the crystals on a macro scale to produce a protein crystal suitable to be analyzed.
The present invention includes a microplate and methods for simultaneously screening a plurality of protein crystallization solutions and producing diffraction quality protein crystals in a vapor-diffusion environment in a high-density high-throughput format.
According to a first aspect of the present invention, there is provided a microplate, comprising a frame including a plurality of wells with defined side-by-side paired chambers of equal size, wherein the side-by-side paired chambers have a maximum volume of about 8 μl, wherein the paired side-by-side chambers have a vapor channel providing vapor exchange between the side-by-side paired chambers.
According to a second aspect of the present invention, there is provided a microplate comprising a frame having a footprint that can be easily handled by a robotic handling system.
According to a third aspect of the present invention, there is provided a microplate, wherein the bottoms of the side-by-side paired chambers are aligned in the same plane.
According to a fourth aspect of the present invention, there is provided a microplate, wherein the bottoms of the side-by-side paired chambers are flat, conical, or concave.
According to a fifth aspect of the present invention, there is provided a microplate, wherein the vapor channel has a predetermined depth and width to allow for a predetermined quantity of a first and second crystallization solution to optimally equilibrate.
According to a sixth aspect of the present invention, there is provided a microplate, wherein the vapor channel is formed by a predetermined opening in a portion of a wall between the side-by-side paired chambers and a transparent adhesive membrane that is positioned over the side-by-side paired chambers.
According to a seventh aspect of the present invention, there is provided a microplate, wherein each well is positioned on said frame such that a liquid handling system can automatically deposit a formulated crystallization solution into one of the side-by-side paired chambers and can automatically deposit a protein solution into the other side-by-side paired chamber.
According to an eighth aspect of the present invention, there is provided a microplate, wherein the high-density high-throughput sitting-drop vapor diffusion protein crystallography microplate has 768 functional wells.
According to a ninth aspect of the present invention, there is provided a microplate, wherein each well is positioned on said frame such that a liquid handling system can automatically deposit the formulated crystallization solution into one of the side-by-side paired chambers and can automatically deposit a protein solution into the other side-by-side paired chamber.
According to a tenth aspect of the present invention, there is provided a method wherein a liquid handling system can automatically deposit a formulated crystallization solution into one of the side-by-side paired chambers of a microplate of the present invention and can automatically deposit a protein solution into the other side-by-side paired chamber of a microplate of the present invention, and wherein the protein solution in one side-by-side paired chamber and the crystallization solution within the second side-by-side paired chamber interact via a vapor diffusion process which enables the formation of protein crystals within the chamber containing the protein solution.
According to an eleventh aspect of the present invention, there is provided a method, wherein the formulated crystallization solutions are selected from the solutions shown in Table 2.
According to a twelfth aspect of the present invention, there is provided a method, wherein the amount of formulated crystallization solution deposited is about 6 μl and the amount of protein solution deposited is about 1 μl.
According to a thirteenth aspect of the present invention, there is provided a method, wherein the amount of formulated crystallization solution deposited is in the range of about 4 μl to about 8 μl and the amount of protein solution deposited is in the range of greater than 0.5 μl to about 2 μl.
A preferred embodiment of the present invention will now be described, by way of an example only, with reference to the accompanying drawings wherein:
Table 1: Stock Components for the 1000 Solution Crystallization Screen: Shown is a table of the stock solution reagent set used to generate the 1000 solution crystallization screen. Stock solutions were either prepared at concentrations based on the solubility information provided in the CRC Handbook of Chemistry or purchased from Hampton Research, Inc.
Table 2: Complete List of 1000 Solutions: Shown is a table listing the composition of all of the 1000 solutions used in the high-density high-throughput screen.
Certain terms are used herein which shall have the meanings set forth as follows.
The term “comprising” means “including principally, but not necessarily solely”. Furthermore, variations of the word “comprising”, such as “comprise” and “comprises”, have correspondingly varied meanings.
The following abbreviations are used herein and throughout the specification:
nl: nanoliter;
μl: microliter;
ml: milliliter;
mm: millimeter;
mg/ml: milligram per millimeter;
° C.: degrees Celsius;
The present invention will now be further described in greater detail. It is to be understood at the outset, that the figures and examples provided herein are to exemplify and not to limit the invention and its various embodiments.
Due to the limited amount of crystallization screens commercially available during the development of the high-throughput crystallization method, a diverse sparse-matrix screen of solutions was designed. Based on the generalization that the crystallization success rate for most proteins is equivalent or greater than 2%, Segelke has suggested that a thorough screen for one protein should consist of approximately 288 crystallization solutions (Segelke 2001). Given the low protein and reservoir requirements of the high-density high-throughput method and microplate of the present invention, it was decided to expand the solution screen to decrease the amount of absent parameter space and improve the chances of producing crystals in a single screen. A 1000 solution screen was developed to cover a crystallization parameter space of approximately 4 times the recommended size discussed by Segelke. In a preferred embodiment, diffraction quality crystals are produced directly from a single 1000 solution screen, but the 1000 solution screen was also designed to provide data on the protein's solubility and information for further optimization of conditions if diffraction quality crystals were not produced during the initial screen.
Ideal components were selected to design a unique 1000 solution screen with a maximum likelihood of generating crystals. Information was gathered from optimum solubility screening articles, the NIST/CARB Biological Macromolecule Crystallization Database, PDB (Brookhaven Protein Data Bank) crystallization parameters, the Hofmeister series, and existing crystallization screens from Hampton Research and Emerald Biosystems (Jancarik and Kim 1991; Saridakis and Chayen 2000). The selected chemicals consisted of 50 precipitants, 12 buffers with alternating pH values, 51 additives, and 8 detergents (Table 1). These chemicals were correlated and entered into the CRYStool™ program (Jena Bioscience GmbH, Germany) to randomly generate 1000 unique solutions. The CRYStool™ program was chosen since it had the capability of producing a screen based on random sampling (Segelke 2001). This reagent set was transferred to a spreadsheet and used to calculate stock reagent concentrations. Selected components were manually combined to create each unique crystallization solution comprising the 1000 solution screen listed in Table 2. The complete set of 1000 solutions is a truly diverse set of solutions with a range of pH, buffers, salts, polymers, alcohols, detergents, and other additives. All of the solutions were prepared in 50 ml conical tubes and transferred into Matrix 96-well deep-well storage blocks (Catalogue #4211, Thermo Fisher Scientific, New Hampshire, USA) for storage at 4° C. Solutions in the deep-well blocks have a shelf life of approximately 1 year.
A microplate and method were needed to quickly set up and use the 1000 solution screen. Although there are alternative methods available, as many as 95% of all crystallization experiments are set up under a vapor diffusion environment. The traditional vapor diffusion method routinely used for more than 20 years utilizes a 24-well deep-well Linbro plate and a suspended 2 μl protein droplet on a glass coverslip. The protein droplet is typically comprised of a 1:1 ratio of protein to crystallization solution and the drop is suspended over 1 ml of crystallization solution. The vapor diffusion method allows the protein droplet to equilibrate with the crystallization solution with water being extracted from the droplet. As the water is extracted during equilibration, the protein and precipitant concentrations slowly increase in the droplet and thus conditions vary over a broad range to promote nucleation and/or crystal growth. Unfortunately the traditional hanging-drop method using 24-well deep-well Linbro plates and a suspended 2 μl protein droplet on a glass coverslip is an extremely laborious and tedious process. In addition, if conventional 24-well Linbro plates were used to conduct the 1000 solution screen, it would have required 42 plates that would have occupied approximately two cubic feet of incubator space, consumed 1 liter of crystallization solutions by using 1 ml of each crystallization solution per well, and taken approximately 16 hours for experimental set up. A 96-well crystallization plate approach would have reduced the number of plates to 11, decreased the total crystallization solution volume to 80 ml by using 80 μl of each crystallization solution per well, and reduced the time to set up the 1000 solution screen to approximately 3 hours.
The present invention provides a microplate and methods to perform sitting-drop vapor diffusion experiments in modified 1536-well Hibase, clear, polystyrene, flat bottom microplates, with 768 functional wells (
Starting from the left side of the microplate, column 1 and every odd column following are designated for well solutions (W) (
The 1000 crystallization solutions are transferred from Matrix 96-well deep-well storage blocks (Catalogue #4211, Thermo Fisher Scientific, New Hampshire, USA) using a Gilson C250 robot (Gilson, Inc., Middleton, Wis., USA) into three 384-well daughter plates (Greiner America, Inc., Catalogue #781201). Each daughter plate is made to contain 80 μl per well of one of the 1000 crystallization solutions. Each daughter plate can accommodate a high-throughput screening cycle of 12 proteins before re-dispensation is necessary. The daughter plates are used to dispense the crystallization solutions into the screening microplates. Two modified 1536-well modified microplates with 768 functional wells are required to run a full screen of 1000 solutions. A first microplate is made to contain 768 experiments in 768 functional wells. A second microplate is made to contain the remaining 232 experiments in 232 functional wells with an additional 536 functional wells for expansion of the screen in the future if more solutions are desired.
To add crystallization solutions and protein solutions to the high-density high-throughput 768 functional well screening microplates, a highly reproducible crystallization routine was developed using the VPrep® automated liquid handling system with a fixed 384 syringe head (Velocity 11, Inc., California, USA). In a typical high-density high-throughput screen, the (W) well receives 6 μl of one of the 1000 crystallization solutions from a 384-well daughter plate and the (P) well receives 0.5 μl of stock protein solution and 0.5 μl of one of the 1000 crystallization solutions for a final volume 1 μl. The crystallization solution used in a 1:1 ratio in each protein droplet well (P) is the same as the corresponding crystallization solution used in each side-by-side paired crystallization solution well (W). After setting up the screening microplate, each well solution (W) has a protein droplet (P) adjacent to it at essentially half the concentration of the crystallization solution (
In order to increase both the throughput and precision necessary to evaluate experiments in the high-density high-throughput 768 functional well microplates, an automated Nikon M3 inverted microscope, Phase 3 Imaging XY stage, and an Evolution MP 5.1 Mega-pixel CCD color camera were assembled to capture and record images. The primary focus was to identify crystals for harvesting and analysis by x-ray diffraction or to identify crystallization leads for data analysis and further optimization to enhance crystal quality. Every captured image, 100 KB per frame, is time date stamped and binned in appropriate folders to create a unique figure array for visualization. It takes approximately 1½ hours to image a complete 1000-well experimental set.
Each set of 1000 images uses approximately 100 MB of disk space and is stored in an internal database to be accessed for comparative examination. The Crystal Evaluator browser, designed in-house, is used to load a set of images and visualize each image. Internal control settings include zoom in/out and light intensity filters to assist with accurate scoring. The scoring process is currently done manually, but can be easily adapted into an automated process once image recognition software becomes further automated. Each image is manually scored against an ordinal 20 number ratings schema to define the visual characteristics of the protein crystallization droplet (
The 1000 solution set and the high-density high-throughput 768 functional well microplate format and method were initially tested using a 15 mg/ml lysozyme stock solution. The test produced a 17.5% hit rate by identifying 175 unique solutions as leads for crystallizing lysozyme. The hits ranged from crystal showers to crystals larger than 0.5 mm. Crystals, ranging from 0.05 mm to greater than 0.5 mm, comprised 14% of the 1000 solutions, with 2% larger than 0.25 mm. The results confirmed that the 1000 solution set and the high-density high-throughput 768 functional well microplate format and method were suitable for generating protein crystals in a screen and for identifying leads for further optimization and crystal generation.
The 1000 solution set and the high-density high-throughput 768 functional well microplate format and method have become invaluable for the process of rapidly screening proteins to identify leads and produce crystals suitable for structure based drug design. Over the past three years, the process has identified 684 leads resulting in the structure determination of 33 proteins or inhibitor complexes from 13 of the 46 therapeutic targets investigated. Surface response data on proteins from all therapeutic areas against each of the 1000 solutions is currently being collected to build a repository for the calculation and prediction of optimal crystallization conditions for unknown proteins.
This application claims priority to Application No. 60/983,960 filed on Oct. 31, 2007, the entire contents of which are incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 60983960 | Oct 2007 | US |
