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Volume 8
Journal of Biotechnology & Biomaterials
ISSN: 2155-952X
Biotech Congress 2018 & Enzymology 2018
March 05-07, 2018
JOINT EVENT
20
th
Global Congress on
Biotechnology
3
rd
International Conference on
Enzymology and Molecular Biology
&
March 05-07, 2018 London, UK
Design, construction and extracellular expression of L-asparaginase from
Dickeya chrysanthemi
in
yeast
Brian Effer
1,2
, Jorge Farias
1
and
Gisele Monteiro
2
1
University of La Frontera, Chile
2
University of São Paulo, Brazil
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-ASNase’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). SuperMan
5
yeast strain was transformed with pJAG-s-
asnB
constructs in order to be able to express the
recombinant protein. Aspartic acid β-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.
breeiky@hotmail.com; b.effer01@ufromail.clBrian Effer et al., J Biotechnol Biomater 2018, Volume 8
DOI: 10.4172/2155-952X-C2-092