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Protein engineering and comparison of microbial expression systems of the biopharmaceutical L-asparaginase

Grant number: 15/07749-2
Support type:Regular Research Grants
Duration: August 01, 2015 - July 31, 2018
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal researcher:Gisele Monteiro
Grantee:Gisele Monteiro
Home Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

L-asparaginase (L-ASNase) is a biopharmaceutical widely used in treating leukemia, especially in Brazil. However, the available commercial formulations are comprised of proteins from bacterial origin, present high molecular weight and they are active only when tetrameric. Thus, problems such as uniformity in drug formulation, allergic reactions and silent inactivation of the enzyme by antibodies binding or serum clearance are found in relation to this biopharmaceutical. This project proposes obtaining micro-organisms that over-express L-ASNase enzyme with improved characteristics by analyzing the effect of the inclusion of post-translational modifications such as glycosylation. To achieve this goal, the strategies will be followed: cloning of L-ASNases in eukaryotic expression system, characterization and comparison of activities and stabilities of these enzymes with and without post-translational modification. The asparaginases chosen for group studies are from bacterial Escherichia coli and Erwinia chrysanthemi. The eukaryotic system that will be tested are conventional strain of Pichia pastoris (GS115) and the Glycoswitch system (P. pastoris strain of genetically modified that performs humanized glycosylation). Only the most promising isoforms in relation to the kinetic characteristics will be evaluated in parallel to their antitumor activity and stability in the presence of human serum in vitro. Insertion of humanized post-translational modifications might be a promising strategy for masking stimulatory epitopes of the human immune system and sites of cysteine-proteases already identified as responsible for the serum clearance in patients. Thus, artificial glycosylation sites may be inserted and evaluated with promising improvement of the biopharmaceutical characteristics. (AU)

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Scientific publications (13)
(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)
WLODARCZYK, SAMARINA R.; CUSTODIO, DEBORA; PESSOA JR, ADALBERTO; MONTEIRO, GISELE. Influence and effect of osmolytes in biopharmaceutical formulations. EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, v. 131, p. 92-98, . (15/07749-2, 13/08617-7, 13/24024-6, 15/24463-5)
WLODARCZYK, SAMARINA R.; COSTA-SILVA, TALES A.; PESSOA-JR, ADALBERTO; MADEIRA, PEDRO; MONTEIRO, GISELE. Effect of osmolytes on the activity of anti-cancer enzyme L-Asparaginase II from Erwinia chrysanthemi. Process Biochemistry, v. 81, p. 123-131, . (13/24024-6, 15/07749-2)
EFFER, BRIAN; LIMA, GUILHERME MEIRA; CABARCA, SINDY; PESSOA, ADALBERTO; FARIAS, JORGE G.; MONTEIRO, GISELE. L-Asparaginase from E. chrysanthemi expressed in glycoswitch: effect of His-Tag fusion on the extracellular expression. PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, v. 49, n. 7, . (17/20384-9, 16/25896-5, 15/07749-2, 13/08617-7, 16/15787-4)
CAVALCANTE, LUCAS DE SOUSA; COSTA-SILVA, TALES A.; SOUZA, TIAGO ANTONIO; IENNE, SUSAN; MONTEIRO, GISELE. Chemogenomic study of gemcitabine using Saccharomyces cerevisiae as model cell-molecular insights about chemoresistance. Brazilian Journal of Microbiology, v. 51, n. 2, p. 489-496, . (11/04938-8, 18/15104-0, 09/01303-1, 15/07749-2)
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, . (13/08617-7, 18/15104-0, 14/16424-7, 18/25994-2, 14/19793-3, 15/07749-2)
COSTA, IRIS MUNHOZ; SCHULTZ, LEONARDO; BIANCHI PEDRA, BEATRIZ DE ARAUJO; MOREIRA LEITE, MARIANA SILVA; FARSKY, SANDRA H. P.; DE OLIVEIRA, MARCOS ANTONIO; PESSOA, ADALBERTO; MONTEIRO, GISELE. Recombinant L-asparaginase 1 from Saccharomyces cerevisiae: an allosteric enzyme with antineoplastic activity. SCIENTIFIC REPORTS, v. 6, . (13/16685-2, 13/08617-7, 15/07749-2)
SANTOS, JOAO H. P. M.; COSTA, IRIS M.; MOLINO, JOAO V. D.; LEITE, MARIANA S. M.; PIMENTA, MARCELA V.; COUTINHO, JOAO A. P.; PESSOA, JR., ADALBERTO; VENTURA, SONIA P. M.; LOPES, ANDRE M.; MONTEIRO, GISELE. Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host. BIOTECHNOLOGY PROGRESS, v. 33, n. 2, p. 416-424, . (15/07749-2, 13/08617-7)
DE SOUSA, GRAZIELE FONSECA; LIMA, MAIRA DE ASSIS; CUSTODIO, DEBORA FERNANDES; FREITAS, VANESSA MORAIS; MONTEIRO, GISELE. Chemogenomic Study of Carboplatin in Saccharomyces cerevisiae: Inhibition of the NEDDylation Process Overcomes Cellular Resistance Mediated by HuR and Cullin Proteins. PLoS One, v. 10, n. 12, . (09/01303-1, 15/07749-2, 11/04173-1)
RODRIGUES, MARIANE A. D.; PIMENTA, MARCELA V.; COSTA, IRIS M.; ZENATTI, PRISCILA P.; MIGITA, NATACHA A.; YUNES, JOSE A.; RANGEL-YAGUI, CARLOTA O.; DE SA, MATHEUS M.; PESSOA, ADALBERTO; COSTA-SILVA, TALES A.; et al. Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase. Biochemical Pharmacology, v. 182, . (14/06863-3, 18/18257-1, 13/08617-7, 16/25896-5, 15/07749-2, 18/15104-0, 13/08139-8, 18/15549-1)
MARCELA VALENTE PIMENTA; GISELE MONTEIRO. The production of biopharmaceuticals in Brazil: current issues. Brazilian Journal of Pharmaceutical Sciences, v. 55, . (15/07749-2)
COSTA, IRIS MUNHOZ; MOURA, DEBORA CUSTODIO; LIMA, GUILHERME MEIRA; PESSOA, ADALBERTO; DOS SANTOS, CAMILA ORESCO; DE OLIVEIRA, MARCOS A.; MONTEIRO, GISELE. Engineered asparaginase from Erwinia chrysanthemi enhances asparagine hydrolase activity and diminishes enzyme immunoreactivity - a new promise to treat acute lymphoblastic leukemia. JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, v. 97, n. 1, . (16/25896-5, 15/07749-2, 18/15104-0, 13/08617-7)
BRUMANO, LARISSA PEREIRA; SANTOS DA SILVA, FRANCISCO VITOR; COSTA-SILVA, TALES ALEXANDRE; APOLINARIO, ALEXSANDRA CONCEICAO; PICADO MADALENA SANTOS, JOAO HENRIQUE; KLEINGESINDS, EDUARDO KREBS; MONTEIRO, GISELE; RANGEL-YAGUI, CARLOTA DE OLIVEIRA; BENYAHIA, BRAHIM; PESSOA JUNIOR, ADALBERTO. Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles. FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, v. 6, . (14/10456-4, 15/07749-2, 18/03734-9, 13/08617-7, 17/21819-9)
LIMA, GUILHERME MEIRA; EFFER, BRIAN; BIASOTO, HENRIQUE PELLIN; FEIJOLI, VERONICA; PESSOA, ADALBERTO; PALMISANO, GIUSEPPE; MONTEIRO, GISELE. Glycosylation of L-asparaginase from E. coli through yeast expression and site-directed mutagenesis. Biochemical Engineering Journal, v. 156, . (15/07749-2, 13/08617-7, 14/06863-3, 18/18257-1, 18/15549-1, 16/15787-4)

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