Abstract
All researchers immersed in the world of recombinant protein production are in agreement that often the production and purification process of a protein can become a nightmare due to an unexpected behavior of the protein at different protocol stages. Once the protein is purified, scientists know that they still cannot relax. There is a decisive last step missing: performing a protein dialysis in a suitable buffer for subsequent experimental trials. Here is when we can find proteins that precipitate during dialysis by buffer-related factors (ionic strength, pH, etc.), which are intrinsic to each protein and are difficult to predict. How can we find the buffer in which a protein is more stable and with less tendency to precipitate? In this chapter we go over possible factors affecting the protein precipitation tendency during the dialysis process and describe a general dialysis protocol with tricks to reduce protein aggregation. Furthermore, we propose a fast method to detect the most appropriate buffer for the stability of a particular protein, performing microdialysis on a battery of different buffers to measure afterwards precipitation by a colorimetric method, and thus being able to choose the most suitable buffer for the dialysis of a given protein.
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Acknowledgment
This work was supported by FIS (PI12/00327) to E.V. and from the Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina (NANOPROVIR project), financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. U.U. received a PhD fellowship from ISCIII and M.P. received a PhD fellowship from UAB (IPF). The authors are also indebted to the Protein Production Platform (CIBER-BBN—UAB) for helpful technical assistance (http://www.bbn.ciber-bbn.es/programas/plataformas/equipamiento).
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Pesarrodona, M., Unzueta, U., Vázquez, E. (2015). Dialysis: A Characterization Method of Aggregation Tendency. In: García-Fruitós, E. (eds) Insoluble Proteins. Methods in Molecular Biology, vol 1258. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2205-5_18
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DOI: https://doi.org/10.1007/978-1-4939-2205-5_18
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Publisher Name: Humana Press, New York, NY
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