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 Opportunities: Regular Research Grants
• Start date: August 01, 2015
• End date: January 31, 2019
• Field of knowledge: Engineering - Chemical Engineering - Chemical Technology
• Agreement: Fundação para a Ciência e a Tecnologia (FCT)
• Principal Investigator: Sandro Roberto Valentini
• Grantee: Sandro Roberto Valentini
• Principal researcher abroad: João Manuel da Costa e Araújo Pereira Coutinho
• Institution abroad: Universidade de Aveiro (UA), Portugal
• Host Institution: Faculdade de Ciências Farmacêuticas (FCFAR). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
• Associated researchers: Adalberto Pessoa Junior ; Jorge 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

(18)

(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)

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. (14/16424-7, 14/19793-3)

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. (14/19793-3, 14/16424-7)

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, p. 19-pg., 2018-07-01. (14/16424-7, 14/01580-3, 16/07529-5, 13/08617-7, 14/19793-3)

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. (15/50058-0, 14/16424-7, 14/19793-3)

MUSSAGY, CASSAMO U.; SANTOS-EBINUMA, VALERIA C.; KURNIA, KIKI A.; DIAS, V, ANA C. R.; CARVALHO, PEDRO; COUTINHO, JOAO A. P.; PEREIRA, JORGE F. B.. Integrative platform for the selective recovery of intracellular carotenoids and lipids from Rhodotorula glutinis CCT-2186 yeast using mixtures of bio-based solvents. GREEN CHEMISTRY, v. 22, n. 23, p. 8478-8494, DEC 7 2020. (14/16424-7, 15/50058-0, 14/19793-3, 17/50303-0)

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. (14/19793-3)

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. (14/16424-7, 14/19793-3, 16/07529-5)

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. (14/16424-7, 14/01580-3, 14/19793-3, 13/08617-7, 16/07529-5)

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, p. 1016-1027, APR 2020. (13/08617-7, 18/15104-0, 14/16424-7, 18/25994-2, 14/19793-3, 15/07749-2)

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. (14/16424-7, 18/50009-8, 18/06576-5, 18/01858-2, 14/19793-3, 16/07529-5)

TORRES-ACOSTA, MARIO A.; DOS SANTOS, V, NATHALIA; VENTURA, SONIA P. M.; COUTINHO, JOAO A. P.; RITO-PALOMARES, MARCO; PEREIRA, JORGE F. B.. Economic analysis of the production and recovery of green fluorescent protein using ATPS-based bioprocesses. Separation and Purification Technology, v. 254, JAN 1 2021. (14/16424-7, 14/19793-3, 16/07529-5)

MUSSAGY, CASSAMO USSEMANE; CARIZIA GUIMARAES, ANA ALICE; FERREIRA ROCHA, LARA VICENTE; WINTERBURN, JAMES; SANTOS-EBINUMA, VALERIA DE CARVALHO; BRANDAO PEREIRA, JORGE FERNANDO. Improvement of carotenoids production from Rhodotorula glutinis CCT-2186. Biochemical Engineering Journal, v. 165, JAN 15 2021. (17/50303-0, 14/16424-7, 14/01580-3, 14/19793-3, 19/09618-3)

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. (17/50303-0, 14/16424-7, 14/01580-3, 14/19793-3)

MUSSAGY, CASSAMO U.; PEREIRA, JORGE F. B.; DUFOSSE, LAURENT; RAGHAVAN, VIJAYA; SANTOS-EBINUMA, VALERIA C.; PESSOA JR, ADALBERTO. Advances and trends in biotechnological production of natural astaxanthin by Phaffia rhodozyma yeast. CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION, AUG 2021. (17/50303-0, 15/50058-0, 14/19793-3, 14/16424-7, 18/06908-8, 20/08655-0)

DOS SANTOS, NATHALIA VIEIRA; SAPONI, CAROLINA FALASCHI; RYAN, TIMOTHY M.; PRIMO, FERNANDO L.; GREAVES, TAMAR L.; PEREIRA, JORGE F. B.. Reversible and irreversible fluorescence activity of the Enhanced Green Fluorescent Protein in pH: Insights for the development of pH-biosensors. International Journal of Biological Macromolecules, v. 164, p. 3474-3484, DEC 1 2020. (18/20833-0, 14/16424-7, 14/19793-3, 18/06576-5, 16/07529-5, 18/50009-8, 18/01858-2)

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. (14/16424-7, 18/50009-8, 14/19793-3, 16/07529-5)

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. (14/16424-7, 14/01580-3, 14/19793-3, 17/50303-0)

V. VERISSIMO, NATHALIA; SAPONI, CAROLINA F.; RYAN, TIMOTHY M.; GREAVES, TAMAR L.; PEREIRA, JORGE F. B.. Imidazolium-based ionic liquids as additives to preserve the Enhanced Green Fluorescent Protein fluorescent activity. GREEN CHEMICAL ENGINEERING, v. 2, n. 4, p. 11-pg., 2021-12-01. (18/06576-5, 18/25511-1, 14/16424-7, 20/14144-8, 18/50009-8, 14/19793-3, 18/01858-2, 18/20833-0, 16/07529-5)