Systematic investigation of protein phase behavior with a microfluidic formulator

Abstract

We demonstrated a microfluidic device for rapidly generating complex mixtures of 32 stock reagents in a 5-nl reactor. This ''formulation chip'' is fully automated and allows thousands of experiments to be performed in a single day with minimal reagent consumption. It was applied to systematically study the phase behavior of the protein xylanase over a large and complex chemical space. For each chemical formulation that demonstrated a pronounced effect on solubility, the protein phase behavior was completely mapped in the chip, generating a set of empirical phase diagrams. This ab initio phase information was used to devise a rational crystallization screen that resulted in 72-fold improvement in successful crystallization hits compared with conventional sparse matrix screens. This formulations tool allows a physicsbased approach to protein crystallization that may prove useful in structural genomics efforts.T he application of x-ray crystallography to the determination of protein structure with atomic resolution was a triumph of structural biology in the 20th century. Since the first solution of the structure of myoglobin in 1958 (1), Ͼ23,000 different structures have been deposited in the Protein Data Bank, and their role in relating structure to function in biology has been profound. As structure determination efforts continue to move past the most tractable crystallization targets (typically small soluble proteins) and focus instead on more challenging macromolecules, such as large protein complexes and membrane proteins (2), the need to better understand and explore the crystallization process has become urgent. That is because once high-quality crystals are in hand, advances in x-ray sources, computer codes, and related technology have made it relatively straightforward to obtain the structure. Determining the appropriate crystallization conditions has become one of the most significant remaining bottlenecks to structure determination (3).Understanding the phase behavior of proteins is an essential part of the crystallization process. The growth of crystals from a protein solution requires the existence of a nontrivial phase diagram, which allows the protein state to be manipulated between at least two thermodynamic phases: soluble and precipitated. The processes of crystal nucleation and growth arise on the boundary between these two phases and are governed by subtle effects in physical chemistry. There are a variety of schemes that manipulate the kinetics of the crystallization process, and all take advantage of generic features of these phase diagrams (4). However, in practice, the phase behavior of very few proteins has been studied in detail (5-12), and solubility information for a specific protein is rarely available for crystallization and optimization experiments (13,14).Furthermore, it is often an arduous process to find the right combination of chemicals that yields appropriate phase behavior for a given protein. Every protein is different, and even a modest subset of stock precipitating solutions...

Mixing with bubbles: a practical technology for use with portable microfluidic devices

“…The creation and integration of these components represents a more substantial problem than it might seem, but the development of new microfluidic elements, and means to incorporate them into devices, are proceeding. Systems for bioanalysis and separation, [1][2][3] or high-throughput screening 4,5 are already available. We are especially interested in devices to be used in resource poor environments-e.g., in healthcare in developing countries, by first responders and the military, and in analogous problems-where portability and ruggedness are key concerns.…”

In situ assembly of linked geometrically coupled microdevices

“…It is well-known and a significant area of research in microfluidics that mixing in microscale geometries is difficult because of the laminar nature of the fluid flows, the associated lack of turbulence, and the resulting reliance on diffusion (21,22). One design that has been used to mix in relatively large microfluidic devices is to use multilayer soft lithography to create pumps and valves and mix fluids by circulating them within a circular channel (23), an approach that has been used, for example, in screening for protein crystallization (24). This technique, however, relies on the supporting pressurization equipment associated with operating many of these pumps simultaneously that limits very high-level parallelization.…”

Enabling the discovery of computational characteristics of enzyme dynamics

“…Microfluidic devices containing hundreds of valves in close proximity were realised [25]. Devices designed to perform complex protocols such as the automatic processing of nucleic acids [26], the generation of complex mixtures for use in on-chip protein experiments [27] and the synthesis of chemical compounds on-chip [28] were demonstrated. Moreover it was shown that device designs containing repeated elements could conduct experiments in parallel [29].…”