Expression of L-asparaginase from Erwinia chrysanthemi in cell-free protein synthesis technology

• Grant number: 18/15041-8
• Support Opportunities: Scholarships in Brazil - Master
• Effective date (Start): November 01, 2018
• Effective date (End): September 30, 2020
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Acordo de Cooperação: Coordination of Improvement of Higher Education Personnel (CAPES)
• 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
• Associated scholarship(s): 19/09354-6 - Directed evolution of L-asparaginase for improving in vivo stability, BE.EP.MS

Abstract

L-Asparaginase (L-ASNase) is an important biopharmaceutical used for the treatment of acute lymphoblastic leukemia. Since L-ASNase is a biological product isolated from bacteria, extensive purification steps are necessary to obtain a pure preparation, which in turn, negatively impacts the costs related to its manufacturing. More economical and efficient alternatives to obtain this bioproduct are therefore required. In this context, we propose to evaluate the possibility to use cell-free protein synthesis systems in order to obtain a pure Erwinia chrysanthemi recombinant L-ASNase. The technology consists in the use of cellular extracts and energetic substrates capable of expressing recombinant proteins in vitro, thus dispensing the use of living cells in the process. The technology has demonstrated high protein expression efficiency, it is scalable to different reaction volumes and the open environment allows for greater process control and immediate evaluation of protein expression levels achieved. The expression of L-ASNase in this technology would simplify its biotechnological process and facilitate the study and characterization of novel forms of the enzyme, in addition to contributing to the improvement and advancement of cell-free protein synthesis technology within the biopharmaceutical industry.

Scientific publications

(6)

(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)

LIMA, GUILHERME M.; MENEZES, MILENE C.; COSTA, IRIS M.; SERRANO, SOLANGE M. T.; MONTEIRO, GISELE. Development of a cell-free protein synthesis protocol to rapidly screen L-asparaginase proteoforms by enzymatic activity. JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, JUN 2021. (13/08617-7, 18/15041-8, 16/25896-5, 13/07467-1, 18/15104-0)

LIMA, GUILHERME M.; MENEZES, MILENE C.; COSTA, IRIS M.; SERRANO, SOLANGE M. T.; MONTEIRO, GISELE. Development of a cell-free protein synthesis protocol to rapidly screen L-asparaginase proteoforms by enzymatic activity. JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, v. 96, n. 9, p. 2659-2666, SEP 2021. (18/15041-8, 13/08617-7, 13/07467-1, 18/15104-0, 16/25896-5)

PARIZOTTO, LETICIA DE ALMEIDA; KLEINGESINDS, EDUARDO KREBS; PEDROTTI DA ROSA, LUIZA MANFRINATO; EFFER, BRIAN; LIMA, GUILHERME MEIRA; HERKENHOFF, MARCOS EDGAR; LI, ZHAOPENG; RINAS, URSULA; MONTEIRO, GISELE; PESSOA, ADALBERTO; et al. Increased glycosylated L-asparaginase production through selection of Pichia pastoris platform and oxygen-methanol control in fed-batches. Biochemical Engineering Journal, v. 173, SEP 2021. (17/20384-9, 18/15041-8, 17/25065-9, 13/08617-7, 19/02583-0, 19/02657-3)

COSTA-SILVA, T. A.; COSTA, I. M.; BIASOTO, H. P.; LIMA, G. M.; SILVA, C.; PESSOA, A.; MONTEIRO, G.. Critical overview of the main features and techniques used for the evaluation of the clinical applicability of L-asparaginase as a biopharmaceutical to treat blood cancer. BLOOD REVIEWS, v. 43, p. 17-pg., 2020-09-01. (18/15041-8, 13/08617-7, 16/25896-5, 15/07749-2)

LIMA, GUILHERME M.; ATRAZHEV, ALEXEY; SARKAR, SUSMITA; SOJITRA, MIRAT; REDDY, REVATHI; TORRES-OBREQUE, KARIN; RANGEL-YAGUI, CARLOTA DE OLIVEIRA; MACAULEY, MATTHEW S.; MONTEIRO, GISELE; DERDA, RATMIR. DNA-Encoded Multivalent Display of Chemically Modified Protein Tetramers on Phage: Synthesis and in Vivo Applications. ACS Chemical Biology, v. 17, n. 11, p. 12-pg., 2022-11-18. (19/09354-6, 16/22065-5, 18/15041-8, 13/08617-7, 18/15104-0)

LIMA, GUILHERME M.; ATRAZHEV, ALEXEY; SARKAR, SUSMITA; SOJITRA, MIRAT; REDDY, REVATHI; TORRES-OBREQUE, KARIN; RANGEL-YAGUI, CARLOTA DE OLIVEIRA; MACAULEY, MATTHEW S.; MONTEIRO, GISELE; DERDA, RATMIR. DNA-Encoded Multivalent Display of Chemically Modified Protein Tetramers on Phage: Synthesis and in Vivo Applications. ACS Chemical Biology, DEC 2021. (13/08617-7, 16/22065-5, 18/15041-8, 19/09354-6, 18/15104-0)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

LIMA, Guilherme Meira. L-asparaginase from Erwinia chrysanthemi: cell-free protein expression and bioconjugation to bacteriophages. 2020. Master's Dissertation - Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) São Paulo.