Cloning of L-asparaginase from Escherichia coli in a Pichia Pastoris strain with humanized glysosylation

• Grant number: 16/15787-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2016
• End date: June 30, 2018
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: Gisele Monteiro
• Grantee: Guilherme Meira Lima
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical, AP.TEM

Abstract

Currently, acute lymphoblastic leukemia (ALL) is the most frequent type of cancer seen in children. One of the main types of treatments available recently is the administration of L-asparaginase (L-ASNase). This enzyme presents itself in different dosage forms in the market, distinguishing within each other either by its source (Escherichia coli ou Erwinia chrysanthemi) or by chemical modifications. However, many of those biopharmaceuticals have some disadvantages, since they may induce some immunogenic reactions and be rapidly cleared from our organism by different elimination processes. Based on this, the focal point of our project is to express L-ASNase enzyme with a glycosylated structure, aiming a less responsive immune reaction and increase in its therapeutical effect. For this, we intend to clone L-ASNase from Escherichia coli in eukaryotic organisms, more precisely yeasts from a genetic modified strain of Pichia pastoris. The goal of this project is to assess if the strain is capable of producing humanized glycosylated proteins as well as secreting it outer of the cell with high efficiency. Therefore, the glycosylation would facilitate the masking of our enzyme, a strategy that could then allow a more efficient therapeutical effect for our biopharmaceutical protein in order to treat ALL.

Scientific publications

(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)

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, APR 2019. (17/20384-9, 16/25896-5, 15/07749-2, 13/08617-7, 16/15787-4)

EFFER, BRIAN; KLEINGESINDS, EDUARDO KREBS; LIMA, GUILHERME MEIRA; COSTA, IRIS MUNHOZ; SANCHEZ-MOGUEL, IGNACIO; PESSOA, ADALBERTO; SANTIAGO, VERONICA FEIJOLI; PALMISANO, GIUSEPPE; FARIAS, JORGE G.; MONTEIRO, GISELE. Glycosylation of Erwinase results in active protein less recognized by antibodies. Biochemical Engineering Journal, v. 163, p. 11-pg., 2020-11-15. (13/08617-7, 15/07749-2, 17/20384-9, 18/15104-0, 16/15787-4, 16/25896-5)

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, APR 15 2020. (15/07749-2, 13/08617-7, 14/06863-3, 18/18257-1, 18/15549-1, 16/15787-4)