Expressão e caracterização bioquímica de uma L-asparaginase II de Bacillus subtilis expressa em Escherichia coli: estudo preliminar de um potencial biofármaco

Abstract

L-asparaginase type II (L-ASNase II) is a therapeutic protein used in the treatment of acute lymphoid leukemia, which affects especially children. It acts by hydrolyzing L-asparagine, an essential amino acid in the proliferation of leukemic lymphoblasts, which depletes the development of tumor cells. Actually, the main L-ASNase II used in chemotherapy originate from the bacteria Escherichia coli and Erwinia chrysanthemi. However, there are many cases of patients who have presented allergic or hypersensitive reactions to these formulations. Therefore, the search for other sources of L-ASNase II, which provides effective treatment and with fewer side effects, becomes important. The objective of this work was to clone the Bacillus subtilis ansZ gene, which encodes an L-asparaginase II, express heterologous E. coli and purify a recombinant protein, to finally carry out a biochemical characterization and evaluation of the therapeutic properties therapies. To this end, genomic DNA from B. subtilis was used as template for PCR amplification of the ansZ gene, which was cloned into the pET28a vector, a plasmid encoding a N-terminal histidine sequence of the expressed protein. Construction was confirmed by automatic nucleotide sequencing, following an expression of the gene in E. coli Rosetta strain. Recombinant L-ASNase II was completed by His-trap column affinity chromatography and MonoQ column ion exchange. The efficiency of purification was visualized on polyacrylamide gel under denaturant conditions. As a result, amplification and cloning of the ansZ gene in pET28a was attested by PCR from transformed colonies and sequencing confirmed a recombinant DNA without mutations. Analysis of heterologous L-ASNase II expression by SDS-PAGE showed non-gel bands at approximately 40 kDa, appearing from the first hour of expression, progressively continuing up to a fourth hour, which is the predicted size of the recombinant enzyme, according to its primary sequence. Analysis of the cytoplasmic solubility of the enzyme shown that it was expressed predominantly in a soluble form, and no considerable formation of inclusion bodies was found. A purification of the enzyme for satisfaction and its catalytic activity was evaluated by the Nessler method, presenting the maximum activity at pH 8.0. It is concluded that the B. subtilis ansZ gene was cloned and successfully expressed in E. coli Rosetta, a recombinant protein obtained predominantly in the soluble form indicating correct folding and nonaggregate formation. Other biochemical and cytotoxic tests are performed to evaluate the potential use of the enzyme as a biopharmaceutical.