Protein Crystallization in a Microfluidic Contactor with Nafion®117 Membranes.

M Polino,M P Pina,J G Crespo,A L Carvalho,Isabel Coelhoso,M J Romão,J G E Gardeniers,H S Rho,Carla A M Portugal,R Mallada

doi:10.3390/membranes11080549

Abstract

Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.

Highlights

The attainment of high-quality diffracting crystals is still the main limitation in protein crystallography, the most employed method for the determination of the three-dimensional structure of proteins
The structure was solved via molecular replacement (MR)
MR is a method widely used in protein crystallography where the structure of a previously studied protein containing similarities with the structure of the protein under investigation is used as a model for recovering the phase information lost during X-ray diffraction and to resolve the structure [36]

Summary

Introduction

The attainment of high-quality diffracting crystals is still the main limitation in protein crystallography, the most employed method for the determination of the three-dimensional structure of proteins. The diffraction quality is dependent on the internal order of the crystal lattice. When the structure of a new protein needs to be unraveled, an enormous number of pre-formulated conditions are commonly screened, before finding those that may lead to crystals with the quality needed for an accurate crystallography analysis [1]. The creativity of scientists has led to the development of several intricate chip designs (valve-based [2], droplet-based [3], slip chips [4], or centrifugal designs [5]) that allowed for the fast screening of hundreds of process conditions, using only very low amounts of protein [6], in devices that can be mounted in front of an X-ray beam, allowing for diffraction screening without crystal handling [7]

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Conclusion