Evaluation of bacterial strains to L-asparaginase super expression: an antileukemic enzime from Escherichia coli

• Grant number: 16/19245-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): December 01, 2016
• Effective date (End): November 30, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Marcos Antonio de Oliveira
• Grantee: Werner Alfinito Feio de Moura
• Host Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , 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

The acute lymphoblastic leukemia (ALL) is the most common neoplasm on childhood, characterized by affecting lymphoblastic cells. Among the drugs used on its treatment we found the enzyme L-Asparaginase (ASNase) from Escherichia coli (EcA). The enzyme is able of hydrolyze the asparagine (Asn) present on the bloodstream, producing aspartic acid (Asp) and NH3. That process reduces the serum levels of Asn, leading to tumor cell apoptosis. The native enzyme is obtained through traditional methodologies and has low yields, which indicates that the recombinant expression might be an interesting alternative to the biopharmaceutical production. In the other hand, recombinant superexpression often leads to the formation of immunogenic protein aggregates, which is considered one of the most deleterious side effects of the treatment. Despite its wide use, the literature analysis reveals that a comparative systematic approach, aiming to mitigate EcA's protein aggregates formation using different strains, has never been performed. Therefore, the aim of this project is to evaluate comparatively the super expression of EcA using different E. coli strains (ArcticExpress (DE3), BL21 (DE3), BL21 (DE3) C43, BL21 Crystal T7 Express, BL21 (DE3) Lemo21, BL21 Origami B, BL21 (DE3) pLysS, BL21 (DE3) Rosetta, K12 SHuffle T7 and BL21 (DE3) Tuner), assessing not only its purity, but also the quantity, the oligomeric state, the aggregation and enzyme activity. The secondary structure will be assessed through circular dichroism spectroscopy (CD), while size exclusion chromatography (SEC) will be used to evaluate the quaternary structure. The enzymatic activity will be performed through Nessler reagent. We believe that those systematic analyses are important for the recombinant biodrug production, and results suitable for publication. (AU)

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)

DE MOURA, WERNER ALFINITO FEIO; SCHULTZ, LEONARDO; BREYER, CARLOS ALEXANDRE; DE OLIVEIRA, ANA LAURA PIRES; TAIRUM, CARLOS ABRUNHOSA; FERNANDES, GABRIELLA COSTA; TOYAMA, MARCOS HIKARI; PESSOA-JR, ADALBERTO; MONTEIRO, GISELE; DE OLIVEIRA, MARCOS ANTONIO. Functional and structural evaluation of the antileukaemic enzymel-asparaginase II expressed at low temperature by differentEscherichia colistrains. Biotechnology Letters, v. 42, n. 11, p. 12-pg., 2020-07-07. (13/08617-7, 11/13500-6, 16/19245-1, 17/25272-4, 19/04054-4, 17/19942-7, 14/22039-9, 17/20291-0)

DE MOURA, WERNER ALFINITO FEIO; SCHULTZ, LEONARDO; BREYER, CARLOS ALEXANDRE; DE OLIVEIRA, ANA LAURA PIRES; TAIRUM, CARLOS ABRUNHOSA; FERNANDES, GABRIELLA COSTA; TOYAMA, MARCOS HIKARI; PESSOA-JR, ADALBERTO; MONTEIRO, GISELE; DE OLIVEIRA, MARCOS ANTONIO. Functional and structural evaluation of the antileukaemic enzymel-asparaginase II expressed at low temperature by differentEscherichia colistrains. Biotechnology Letters, JUL 2020. (13/08617-7, 17/19942-7, 17/25272-4, 19/04054-4, 14/22039-9, 16/19245-1, 17/20291-0, 11/13500-6)