期刊
BIOCHEMICAL ENGINEERING JOURNAL
卷 156, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.bej.2020.107516
关键词
Site-directed mutagenesis; Asparaginase; Glycosylation; Biopharmaceutical; Biologics; Yeast
资金
- Sao Paulo Research Foundation (FAPESP/Brazil) [2013/08617-7, 2015/07749-2, 2016/15787-4]
- Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT/Chile) [21150288]
- Brazilian National Counsel of Technological and Scientific Development [CNPq 309595/2016-9]
- CAPES [001]
- FAPESP [2014/06863-3, 2018/18257-1, 2018/15549-1]
- bolsa de produtividade by CNPq
L-Asparaginase (L-ASNase) is a key component in the treatment of acute lymphoblastic leukemia (ALL), but several clinical disadvantages, such as immunogenicity and rapid clearance, are still present. We evaluated the possibility to synthesize a new L-ASNase from Escherichia coll. with human-like glycosylation and study the glycosylation effect on the biochemical properties of the enzyme. Six L-ASNase mutants were also created in which L-ASNase glycosylation sites were removed through site-directed mutagenesis. The WT L-ASNase was successfully expressed, secreted and glycosylated by an engineered P. pastoris strain and presented predominantly Man 5 G1cNAc 2 glycans on its structure, which were then able to decrease L-ASNase immunogenicity in vitro. The purified glycosylated L-ASNase has shown a 30-fold decrease in specific enzymatic activity compared to the non-glycosylated proteoform, but a triple mutant L-ASNase (3M) was able to restore L-ASNase biological activity to significant levels. 3M accumulated in the yeast periplasmic space and there presented a 28fold increase in enzymatic activity when compared to the fully glycosylated proteoform. Both WT and 3M L-ASNases presented increased stability in human serum compared to non-glycosylated L-ASNase. This study demonstrates the important effects of glycosylation on L-ASNase properties and opens up new possibilities to use glycosylated L-ASNases for the treatment of ALL.
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