Brian Effer
University of La Frontera, Chile
Title: Design, construction and extracellular expression of L-asparaginase from Dickeya chrysanthemi in yeast
Biography
Biography: Brian Effer
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-ASNase’s immunogenic eff ects, it is necessary to seek alternatives such as recombinant expression in yeast. Th is
expression system can provide extracelullar secretion and glycosilation process, which can decrease immunogenicity and
facilitate downstream process. We report the construction of three diff erent 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 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, fi nal enzyme activity and cell secretion.
Purifi cation processes are being executed.