Journal of Biotechnology & Biomaterials
Open Access

Abstract

L-asparaginase (L-ASNase) is an important enzyme used as a biopharmaceutical to treat acute lymphoblastic leukemia (ALL). Currently there are two L-ASNase approved by FDA: native and of bacterial origin, both from E. coli and D. chrysanthemi. Due to L-ASNaseâ��s immunogenic effects, it is necessary to seek alternatives such as recombinant expression in yeast. This expression system can provide extracelullar secretion and glycosilation process, which can decrease immunogenicity and facilitate downstream process. We report the construction of three different expression vectors in order to obtain extracelullar L-ASNase from D. chrysanthemi using eukaryotic exrpression system. asnB gene from D. chrysanthemi was cloned in pJAG-s1 plasmid in fusion with endogenous signal sequence (SS), that addresses protein to bacterial periplasm, and with or without histidine tag (His). SuperMan5 yeast strain was transformed with pJAG-s-asnB constructs in order to be able to express the recombinant protein. Aspartic acid �²-hydroxamate method was applied for activity determination of L-ASNase recombinant in culture supernatants. When both SS and His-tag were removed (expression of mature protein), protein expression and secretion process were improved considerably compared to other constructions, indicating that for this gene, additional structures added to the recombinant protein may interfere with the expression, final enzyme activity and cell secretion. Purification processes are being executed.

Biography

Brian Effer is a PhD candidate at the University of La Frontera and University of São Paulo in Cell and Molecular Biology and Biochemical and Pharmaceutical Technology areas, respectively. He has published seven papers in reputed journals and has been serving as a referee in several journals.
Email:breeiky@hotmail.com; b.effer01@ufromail.cl