Expressão in vitro da enzima l-asparaginase II de Escherichia coli em células de mamíferos

Abstract

L-Asparaginase II is an amidohydrolase used in the treatment of Acute Lymphoblastic Leukemia's treatment (ALL) which catalyzes the hydrolysis of asparagine in aspartate and ammonia. Since tumor cells lacks the enzyme asparagine synthetase, they are not able to synthetize asparagine, thus depending of the amino acid from extracellular sources. However, the therapeutic L-Asparaginase is currently purified or produced in Escherichia coli, what could be potentially linked to the high number of immune responses in patients who undergo treatment and sometimes lead to its discontinuation. These immune responses may be associated with the bacterial origin of the protein, which has epitopes that can trigger hypersensitivity reactions in patients. Mammalian cells, unlike bacterial ones, have the ability to perform post-translational modifications as glycosylations, which may be associated with a more adequate protein conformation and also potentially masking of allergenic epitopes. The aim of this work was to express L-asparaginase in human cells, determine its viability, the occurrence of post-translational modifications and the comparison between the enzyme produced in bacteria and that produced in human cells. Thorough recombinant DNA technology, the in vitro expression of L-Asparaginase in a human embryonic kidney cell line (HEK-293) was successfully performed, with the production two different glycoforms. The enzyme expressed in human cells had maximum activity at pH 8 and temperature 60° C, while L-Asparaginase expressed in E. coli had pH 8.5 and optimal temperature 60°C. The DNA construct generated in this work have applicability to other expression systems, making possible the future comparison of the recombinant protein generated by different biopharmaceutical production platforms.