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Development of a cell-free protein synthesis protocol to rapidly screen L-asparaginase proteoforms by enzymatic activity
J Chem Technol Biotechnol, v. 96, n. 9, p. 2659-2666, set. 2021
Article en En | SES-SP, SESSP-IBPROD, SES-SP | ID: bud-3865
Biblioteca responsable: BR78.1
ABSTRACT
Abstract 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 3.5.1.1, 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. © 2021 Society of Chemical Industry (SCI).
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Texto completo: 1 Colección: 06-national / BR Base de datos: SES-SP / SESSP-IBPROD Tipo de estudio: Guideline Idioma: En Revista: J Chem Technol Biotechnol Año: 2021 Tipo del documento: Article

Texto completo: 1 Colección: 06-national / BR Base de datos: SES-SP / SESSP-IBPROD Tipo de estudio: Guideline Idioma: En Revista: J Chem Technol Biotechnol Año: 2021 Tipo del documento: Article