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Optimization and scale-up of novel Ionic-Liquid-based purification processes for recombinant green fluorescent protein produced by Escherichia coli "GFPurIL"

Grant number: 14/19793-3
Support type:Regular Research Grants
Duration: August 01, 2015 - January 31, 2019
Field of knowledge:Engineering - Chemical Engineering - Chemical Technology
Cooperation agreement: Fundação para a Ciência e a Tecnologia (FCT)
Principal Investigator:Sandro Roberto Valentini
Grantee:Sandro Roberto Valentini
Principal investigator abroad: João Manuel da Costa e Araújo Pereira Coutinho
Institution abroad: Universidade de Aveiro (UA), Portugal
Home Institution: Faculdade de Ciências Farmacêuticas (FCFAR). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
Assoc. researchers:Adalberto Pessoa Junior ; Jorge Fernando Brandão Pereira ; Mara Guadalupe Freire Martins ; Sónia Patrícia Marques Ventura ; Valéria de Carvalho Santos Ebinuma

Abstract

One of the new challenges of Biotechnology and Pharmaceutical industries is the use of proteins as innovative therapies for the treatment of complex diseases. In order to control their production and/or to identify their applicability and eficcacy, the development of new biomarker/bioreporter proteins is required. For that purpose, the strong fluorescence and easy detection of Green Fluorescent Protein (GFP) makes of it one of the most promising biological biomarkers. As for other proteins/enzymes produced by microorganisms, its production and purification is a complex and expensive process, which limits a more extensive use of GFP. This project proposes the use of Ionic Liquids-based Aqueous Two Phase Systems (IL-ATPS) as an alternative and more sustainable methodology to isolate and purify a recombinant GFP produced by Escherichia coli. To accomplish this goal, this project will begin with the expression and production of GFP, and posterior optimization of the purification from fermented broth using four different IL-ATPS approaches. Then, it will carry out the scale-up of the technological platform optimized at Lab Scale to a Pilot Scale. With this project it is expected the development and evaluation of the industrial applicability of a new sustainable downstream technology for production of GFP, which could be then further extended to other high-valued proteins and bioproducts. (AU)

Scientific publications (11)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MUSSAGY, CASSAMO U.; TABANEZ, NAIARA L.; FARIAS, FABIANE O.; KURNIA, KIKI A.; MAFRA, MARCOS R.; PEREIRA, JORGE F. B. Determination, characterization and modeling of aqueous biphasic systems composed of propylammonium-based ionic liquids and phosphate salts. Chemical Physics Letters, v. 754, SEP 2020. Web of Science Citations: 0.
MAGRI, AGNES; PIMENTA, MARCELA V.; SANTOS, JOAO H. P. M.; COUTINHO, JOAO A. P.; VENTURA, SONIA P. M.; MONTEIRO, GISELE; RANGEL-YAGUI, CARLOTA O.; PEREIRA, JORGE F. B. Controlling the l-asparaginase extraction and purification by the appropriate selection of polymer/salt-based aqueous biphasic systems. JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, v. 95, n. 4 DEC 2019. Web of Science Citations: 0.
MUSSAGY, CASSAMO U.; SANTOS-EBINUMA, VALERIA C.; GONZALEZ-MIQUEL, MARIA; COUTINHO, JOAO A. P.; PEREIRA, JORGE F. B. Protic Ionic Liquids as Cell-Disrupting Agents for the Recovery of Intracellular Carotenoids from Yeast Rhodotorula glutinis CCT-2186. ACS SUSTAINABLE CHEMISTRY & ENGINEERING, v. 7, n. 19, p. 16765-16776, OCT 7 2019. Web of Science Citations: 0.
DOS SANTOS, NATHALIA VIEIRA; SAPONI, CAROLINA FALASCHI; GREAVES, TAMAR LOUISE; BRANDAO PEREIRA, JORGE FERNANDO. Revealing a new fluorescence peak of the enhanced green fluorescent protein using three-dimensional fluorescence spectroscopy. RSC ADVANCES, v. 9, n. 40, p. 22853-22858, AUG 1 2019. Web of Science Citations: 0.
LOPES, CAMILA; DOS SANTOS, NATHALIA VIEIRA; DUPONT, JANA; PEDROLLI, DANIELLE BISCARO; VALENTINI, SANDRO ROBERTO; SANTOS-EBINUMA, VALERIA DE CARVALHO; BRANDAO PEREIRA, JORGE FERNANDO. Improving the cost effectiveness of enhanced green fluorescent protein production using recombinant Escherichia coli BL21 (DE3): Decreasing the expression inducer concentration. Biotechnology and Applied Biochemistry, v. 66, n. 4, p. 527-536, JUL 2019. Web of Science Citations: 0.
RODRIGUES TEIXEIRA-PINTO, RENATA GARCIA; MOLINO, JOAO VITOR DUTRA; SANTOS-EBINUMA, VALERIA CARVALHO; PESSOA, JR., ADALBERTO; VALENTINI, SANDRO ROBERTO; BRANDAO PEREIRA, JORGE FERNANDO; LOPES, ANDRE MORENI. Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 2. Nonionic micellar systems. Separation and Purification Technology, v. 210, p. 69-79, FEB 8 2019. Web of Science Citations: 0.
MUSSAGY, CASSAMO USSEMANE; WINTERBURN, JAMES; SANTOS-EBINUMA, VALERIA CARVALHO; BRANDAO PEREIRA, JORGE FERNANDO. Production and extraction of carotenoids produced by microorganisms. Applied Microbiology and Biotechnology, v. 103, n. 3, p. 1095-1114, FEB 2019. Web of Science Citations: 8.
LOPES, ANDRE MORENI; DUTRA MOLINO, JOAO VITOR; DOS SANTOS-EBINUMA, VALERIA CARVALHO; PESSOA, JR., ADALBERTO; VALENTINI, SANDRO ROBERTO; BRANDAO PEREIRA, JORGE FERNANDO. Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 1. Polymer-polymer systems. Separation and Purification Technology, v. 206, p. 39-49, NOV 29 2018. Web of Science Citations: 4.
DOS SANTOS, NATHALIA V.; MARTINS, MARGARIDA; SANTOS-EBINUMA, VALERIA C.; VENTURA, SONIA P. M.; COUTINHO, JOAO A. P.; VALENTINI, SANDRO R.; PEREIRA, JORGE F. B. Aqueous Biphasic Systems Composed of Cholinium Chloride and Polymers as Effective Platforms for the Purification of Recombinant Green Fluorescent Protein. ACS SUSTAINABLE CHEMISTRY & ENGINEERING, v. 6, n. 7, p. 9383-9393, JUL 2018. Web of Science Citations: 6.
DOS SANTOS, NATHALIA VIEIRA; SANTOS-EBINUMA, VALERIA DE CARVALHO; PESSOA JUNIOR, ADALBERTO; BRANDAO PEREIRA, JORGE FERNANDO. Liquid-liquid extraction of biopharmaceuticals from fermented broth: trends and future prospects. JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, v. 93, n. 7, SI, p. 1845-1863, JUL 2018. Web of Science Citations: 9.
NEVES, CATARINA M. S. S.; SHAHRIARI, SHAHLA; LEMUS, JESUS; PEREIRA, JORGE F. B.; FREIRE, MARA G.; COUTINHO, JOAO A. P. Aqueous biphasic systems composed of ionic liquids and polypropylene glycol: insights into their liquid-liquid demixing mechanisms. Physical Chemistry Chemical Physics, v. 18, n. 30, p. 20571-20582, AUG 14 2016. Web of Science Citations: 17.

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