| Full text | |
| Author(s): |
Santos, Joao H. P. M.
;
Costa, Iris M.
;
Molino, Joao V. D.
;
Leite, Mariana S. M.
;
Pimenta, Marcela V.
;
Coutinho, Joao A. P.
;
Pessoa, Jr., Adalberto
;
Ventura, Sonia P. M.
;
Lopes, Andre M.
;
Monteiro, Gisele
Total Authors: 10
|
| Document type: | Journal article |
| Source: | BIOTECHNOLOGY PROGRESS; v. 33, n. 2, p. 416-424, MAR-APR 2017. |
| Web of Science Citations: | 3 |
| Abstract | |
l-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc\_ASNaseI and Sc\_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc\_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc\_ASNaseI (His)(6)-tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni2+-charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc\_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc\_ASNaseI with high specific activity (110.1 +/- 0.3 IUmg(-1)) were obtained. In addition, the use of FPLC-IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 +/- 0.01%) was achieved with a purification factor of 17-fold. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416-424, 2017 (AU) | |
| FAPESP's process: | 15/07749-2 - Protein engineering and comparison of microbial expression systems of the biopharmaceutical L-asparaginase |
| Grantee: | Gisele Monteiro |
| Support Opportunities: | Regular Research Grants |
| 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 Opportunities: | Research Projects - Thematic Grants |