Development of sustainable platforms for the stabilization and purification of the recombinant Green Fluorescent Protein produced by Escherichia coli

Author(s)

Nathalia Vieira dos Santos

• Document type: Doctoral Thesis
• Press: Araraquara. 2020-12-19.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2020-11-26
• Advisor: Jorge Fernando Brandão Pereira; Fernando Lucas Primo

Abstract

Biotechnology allowed the development of novel biomolecules for medical and industrial applications. However, there are still limitations to the use of many outstanding biological products at large-scale due to their low stability (which hinders their distribution, storage, and handling) and very high prices (mostly caused by their intricate and costly production and purification processes). A better understanding of the properties and stability of biomolecules, allied with the development of simpler, cheaper, and more sustainable stabilization and purification platforms for biotechnological products, can help to overcome these limitations and expand their commercial use. Hence, the goal of this work was to improve the large-scale applications of biomolecules using the Enhanced Green Fluorescent Protein (EGFP, an important biomarker and biosensor) as a proof of concept. In this sense, the researcher designed in-depth studies of EGFP structure and fluorescence properties, evaluated its stability in the presence of different solvents or under stress conditions, and developed novel sustainable stabilization and purification systems for EGFP using Ionic Liquids (ILs), organic salts, and polymers compatible with biomolecules. In the studies of EGFP properties and structure, it was possible to detect an additional and never reported fluorescence peak for this protein and solve a scientific controversy regarding EGFP conformations and activity. Additionally, on the pH stability studies, it was possible to demonstrate the pH-dependence of the fluorescence of EGFP and elucidate the impacts of acidic pH on the structure of the protein, which can aid in the development of novel neutral-to-acidic pH-biosensors. The studies of EGFP activity in the presence of ILs and under stress conditions showed ILs have different impacts on the stability of proteins, depending on their nature, concentration, and alkyl chain length. For example, some ILs like cholinium chloride, cholinium octanoate, and cholinium decanoate protected EGFP fluorescence, while imidazolium-based ILs with longer alkyl chain length reduced the activity of the protein. Furthermore, it was possible to find ILs that tripled the long-term preservation of EGFP activity at room temperature, which could potentially act as protein stabilizers in biopharmaceutical formulations. For the purification stage, it was possible to develop a sustainable purification platform associating aqueous biphasic systems (composed of polymers and salts) and ultrafiltration, obtaining > 97 % pure EGFP while recovering 60 % of the solvents used in the process. In conclusion, the study of EGFP activity and stability, allied with the development of sustainable alternative low-cost purification techniques, can broaden its commercial use and have potential applications for other biomolecules. (AU)