<b>Non-Contact Liquid Handling To Perform Simultaneous Microbatch And Vapor Diffusion Crystallization Screens</b><br/><br/>

Non-Contact Liquid Handling To Perform Simultaneous Microbatch And Vapor Diffusion Crystallization Screens

by James E Johnson, Mary Cornett, Ph.D. and Jan Lõwe

Introduction

Protein crystallography utilizes a variety of techniques and is an immensely beneficial tool for investigators in the fields of structure identification, rational synthesis and lead optimization in drug discovery. Appropriate crystallization conditions for a given protein, however, are impossible to predict and hence require the rather arduous task of experimental iteration. This process requires the testing of precious protein solutions in conjunction with a large set of crystal-growing conditions until an appropriate condition set can be found. Unfortunately, this iterative process is one that is prone to irreproducibility and investigator bias resulting from the tendency to use only techniques that have proven successful in the past. This biased approach, however, is often without any quantitative merit and can lead to missed opportunities during the initial screening process.

Figure 1. Innovadyne's Platemaker 96+8 instrument.

Using multiple well-known protein crystallographic techniques, such as microbatch and vapor diffusion simultaneously, has been shown to produce different yet complimentary results. In their application note, A Comparison of Microbatch and Vapor Diffusion for Initial Screening of Crystallization Conditions, Baldock, Mills, and Stewart (Douglas Instruments Ltd., Douglas House, East Garston, Hungerford, Berks, UK) find that there are numerous conditions where crystals are formed uniquely to a technigue when using microbatch and vapor diffusion methods in side-by-side experiments. They cite data that shows that out of a total of 58 conditions generating crystals only 26 conditions were redundantly found by both techniques, with microbatch generating 17 unique "hits" while vapor diffusion produced 15 similarly unique "hits". Using these complimentary techniques together can be a powerful tool for increasing the odds of crystal generation by maximizing the crystallographic conditions under consideration. Simultaneously, having more and different initial hits available can increase chances of obtaining diffraction-quality crystals. However, it is very difficult to generate these simultaneous experiments using a single automated instrument platform. In this paper we will discuss the use of a single non-contact liquid handler, which has the capability of performing both vapor diffusion experiments and microbatch experiments side-by-side. This instrument also allows for optimization of experimental parameters such as the volume and number of protein additions, protein concentrations, and precipitant concentrations.

Background

All methods of crystallization involve a phase transition in which the protein is in solution at the start of the experiment and comes out of the solution to form crystals when the solution is brought into supersaturation. Once nuclei have formed, the concentration of protein in the solute will drop, thereby leading the system into the meta-stable zone where growth should occur without the formation of further nuclei. Alternatively and ideally, crystal nucleation is much slower than crystal growth, leading to drops with few large crystals.^(1,2,3,4,5) Vapor-diffusion methods such as sitting or hanging drop screens involve an aqueous drop containing the protein and the crystallization agents in an amount lower than that required for the formation of crystals. This drop is equilibrated against a reservoir that gradually concentrates the ingredients in the protein drop until equilibrium is reached by the 'vapor-diffusion' of water. During the diffusion process (in which both precipitating agents and the protein become gradually more concentrated), a single crystallization trial proceeds through a range of conditions, thereby conducting a self-screening process.

The objective of the microbatch technique is to reduce the consumption of sample by generating crystallization trials in very small volumes. The samples, which are dispensed and incubated under the surface of a low-density oil, are protected from evaporation, contamination and physical shock by the oil.⁽⁶⁾ The process is similar to vapor diffusion in its use of materials, but very different because the experimental diffusion is limited by the oil, thus constraining the "self screening" characteristic seen in vapor diffusion experiments. Chayen has shown approximate conditions that make one technique transferable to the other. A modification of the method uses a mixture of paraffin and silicone oil to allow for the slow evaporation of drops, thus greatly improving the chances of success by 'self-screening' a range of different concentrations.⁽⁷⁾

Liquid handling advances

Protein crystallography, with the vast array of reagents that are dispensed, requires expert liquid handling to reconcile viscosity differences and minimize protein waste, often at sub-microliter volumes, while performing these tasks within a cycle time that constrains evaporation. Although vapor diffusion experiments were traditionally performed in microliters, advances in sitting drop and hanging drop labware, liquid handling, and detection devices now routinely facilitate sub-microliter volume screens. Small total volumes are extremely important when the target protein is a membrane protein, as there often is a very small amount of protein available. Using simultaneous microbatch and vapor diffusion screens makes it possible to use the small amount of protein available towards generating the most likely set of conditions that will produce a useable crystal.

Because of the small volumes now being used for protein crystallography, it is very difficult to accomplish these experiments by hand. There are any number of automated systems that can perform sitting drop experiments. However, fewer systems can perform hanging drop and microbatch screens, due to the complexity of the liquid handling tasks. The Screenmaker and Platemaker suite of instruments (Innovadyne Technologies, Inc., Santa Rosa, CA, Figure 1) feature independent control of all 104 dispense channels. Both instruments are able to use any tip to dispense to any position of a crystallography plate. This gives unlimited opportunities for the development of screens, gradients, and additives. In addition, with a volume range that goes from 10s of nanoliters to 10s of microliters, a variety of tasks can be accomplished including the transfer of large volumes from a deep-well labware site to a crystal plate, multiple high-speed protein additions, mirror image dispenses, additive additions, and microbatch liquid handling dispensing through oil. Cycle times that minimize evaporation, i.e. <60 s, give the crystallographer the freedom to investigate lower volumes.

Advances in crystallography labware

Figure 2. UO-1 Microbatch Plate.

Newer sitting drop plate designs have greatly advanced the flexibility of screens with several crystal drop chambers available for multiple experiments against a single set of buffer conditions. The MRC Laboratory of Molecular Biology (MRC), under the guidance of Jan Lõwe and in collaboration with a major plate manufacturer, has introduced the SD-2 protein crystallography plate (Innovadyne Technologies). This optically clear plate is produced using a new polymer that reduces through-plastic evaporation, greatly enhances imaging efforts with a lens shape design that improves illumination but also enables liquid handling by effectively focusing the drop in the center of the well. Recently, the MRC has also designed a microbatch plate with similar optical properties and large wells, the UO-1, to be used in microbatch experiments (Innovadyne Technologies, Figure 2). In addition, MRC has developed a protocol using an oil that allows a small amount of evaporation to occur, facilitating the formation of even more crystals than traditional microbatch, and that circumvents the problems associated with traditional silicone/praffin oil mixtures that spread along plastic surfaces. Combined with the Innovadyne Screenmaker/Platemaker instruments that have the capability to dispense to both plates during the same screen, this complete crystallographic screening system gives the crystallographer a much greater probability of successful crystallization screens.

Future work

As membrane protein isolation and expression becomes common, low volume dispensing will become mandatory for structural identification of the proteins through protein crystallography screens. Innovadyne's Platemaker, with the capability of dispensing down to 50 nL to dry plates and through oil, makes it possible to not only run a primary screen on the protein of choice but to also run additional optimized screens that may provide a greater chance of producing a crystal of sufficient quality to determine its structure. Because these proteins are some of the most sought-after targets for drug candidates, the new advance liquid handling capabilities and labware described here may provide a means to better leverage the vast potential of protein crystallography.

James E. Johnson and Mary Cornett, Ph.D., are with Innovadyne Technologies and Jan Lõwe is with the MRC Laboratory of Molecular Biology More information about the technology discussed in this article is available from:

Innovadyne Technologies, Inc.
707-547-2500,
www.innovadyne.com

References

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