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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Development of a cell-free protein synthesis protocol to rapidly screen L-asparaginase proteoforms by enzymatic activity

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Lima, Guilherme M. [1] ; Menezes, Milene C. [2] ; Costa, Iris M. [1] ; Serrano, Solange M. T. [2] ; Monteiro, Gisele [1]
Total Authors: 5
[1] Univ Sao Paulo, Fac Ciencias Farmaceut, Dept Tecnol Bioquim Farmaceut, BR-05508000 Sao Paulo, SP - Brazil
[2] Inst Butantan, Lab Toxinol Aplicada, Ctr Toxins Immune Response & Cell Signaling CeTIC, Sao Paulo - Brazil
Total Affiliations: 2
Document type: Journal article
Web of Science Citations: 0

BACKGROUND Cell-free protein synthesis (CFPS) technology has emerged as a powerful tool for a variety of biotechnological applications, including the expression of different classes of biopharmaceutical products. L-Asparaginase (E.C. Number:, L-asparagine amidohydrolase) (L-ASNase) is an important biopharmaceutical used to treat leukemia, but expression of multiple proteoforms in CFPS systems and rapid characterization using standard colorimetric methods has not yet been fully exploited. Herein, recombinant expression and characterization of an L-ASNase from Erwinia chrysanthemi (Erwinase) using a new CFPS protocol is reported. RESULTS Expression and quantification of the enzymatic activity of a soluble his-tagged L-ASNase directly from a CFPS reaction was successfully achieved. Purification of the protein was not required in order to assess its biological activity. Activity of L-ASNase was significantly higher than the control reaction (7.07 +/- 0.68 U mL(-1) vs. 1.83 +/- 0.14 U mL(-1), respectively). Expression of a mutant Erwinase proteoform - V293M - was also achieved and it presented a similar enzymatic activity. No significant loss in L-ASNase enzymatic activity was noticed after removal of cyclic AMP, spermidine, transfer RNA, T7 RNA polymerase and, especially, ammonium acetate (a common interference in ASNase enzymatic assays) from the CFPS reaction. CONCLUSION The protocol developed in this work will facilitate the screening of novel clinically-relevant L-ASNase proteoforms. (c) 2021 Society of Chemical Industry (SCI). (AU)

FAPESP's process: 18/15104-0 - Preclinical assays of glycoprotein asparaginase proteoforms or resistant to serum proteases.
Grantee:Gisele Monteiro
Support type: Regular Research Grants
FAPESP's process: 18/15041-8 - Expression of L-asparaginase from Erwinia chrysanthemi in cell-free protein synthesis technology
Grantee:Guilherme Meira Lima
Support type: Scholarships in Brazil - Master
FAPESP's process: 16/25896-5 - Biochemical characterization and cytotoxic evaluation of mutant isoforms of L-Asparaginase II from Dickeya chrysanthemi (Erwinia chrysanthemi)
Grantee:Iris Munhoz Costa
Support type: Scholarships in Brazil - Doctorate
FAPESP's process: 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical
Grantee:Adalberto Pessoa Junior
Support type: Research Projects - Thematic Grants
FAPESP's process: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling
Grantee:Hugo Aguirre Armelin
Support type: Research Grants - Research, Innovation and Dissemination Centers - RIDC