Abstract
Production, extraction, purification, and stabilization of integral membrane proteins are key steps for successful structural biology studies, in particular for X-ray crystallography or single particle microscopy. Here, we present the purification protocol of CntI from Pseudomonas aeruginosa, a new metallophore exporter of the Drug Metabolite Transporter (DMT) family involved in pseudopaline secretion. Subsequent to CntI purification, we optimized the buffer pH, salts, and additives by differential scanning fluorimetry (DSF), also known as Thermofluor Assay (TFA) or fluorescent thermal stability assay (FTSA), with the use of dye 1-AnilinoNaphthalene-8-Sulfonic acid (ANS), a fluorescent molecule compatible with detergents. After the buffer optimization, the purified CntI was analyzed by Size Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering (SEC-MALLS), UV absorbance, and Refractive Index detectors, in order to determine the absolute molar mass of the protein–detergent complex, the detergent amount bound to the protein and the amount of protein-free detergent micelles. Altogether, these biophysical techniques give preliminary and mandatory information about the suitability of the purified membrane protein for further biophysical or structural investigations.
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Acknowledgments
We thank Dr. Pascal Arnoux for cloning and providing the expression plasmid of CntI in pET-TEV. We thank Dr. Daniel Picot for his help in peak integration of refractive index curves.
This work was supported by “Agence Nationale de la Recherche,” grant number ANR-20-CE11-0018).
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Mégret-Cavalier, M., Pozza, A., Cece, Q., Bonneté, F., Broutin, I., Phan, G. (2024). Starting with an Integral Membrane Protein Project for Structural Biology: Production, Purification, Detergent Quantification, and Buffer Optimization—Case Study of the Exporter CntI from Pseudomonas aeruginosa. In: Journet, L., Cascales, E. (eds) Bacterial Secretion Systems . Methods in Molecular Biology, vol 2715. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3445-5_26
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DOI: https://doi.org/10.1007/978-1-0716-3445-5_26
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