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Coupling High Throughput Microfluidics and Small-Angle X‑ray Scattering to Study Protein Crystallization from Solution
journal contribution
posted on 2017-01-17, 00:00 authored by Nhat Pham, Dimitri Radajewski, Adam Round, Martha Brennich, Petra Pernot, Béatrice Biscans, Françoise Bonneté, Sébastien TeychenéIn
this work, we propose the combination of small-angle X-ray scattering
(SAXS) and high throughput, droplet based microfluidics as a powerful
tool to investigate macromolecular interactions, directly related
to protein solubility. For this purpose, a robust and low cost microfluidic
platform was fabricated for achieving the mixing of proteins, crystallization
reagents, and buffer in nanoliter volumes and the subsequent generation
of nanodroplets by means of a two phase flow. The protein samples
are compartmentalized inside droplets, each one acting as an isolated
microreactor. Hence their physicochemical conditions (concentration,
pH, etc.) can be finely tuned without cross-contamination, allowing
the screening of a huge number of saturation conditions with a small
amount of biological material. The droplet flow is synchronized with
synchrotron radiation SAXS measurements to probe protein interactions
while minimizing radiation damage. To this end, the experimental setup
was tested with rasburicase (known to be very sensitive to denaturation),
proving the structural stability of the protein in the droplets and
the absence of radiation damage. Subsequently weak interaction variations
as a function of protein saturation was studied for the model protein
lysozime. The second virial coefficients (A2) were determined from
the X-ray structure factors extrapolated to the origin. A2 obtained
values were found to be in good agreement with data previously reported
in literature but using only a few milligrams of protein. The experimental
results presented here highlight the interest and convenience of using
this methodology as a promising and potential candidate for studying
protein interactions for the construction of phase diagrams.