Generation and isolation of recombinant DNase II enzyme

Adrian Alberto Mejia Lara, University of Texas at El Paso

Abstract

Deoxyribonuclease II (DNaseII) is an endonuclease that functions optimally at acid pH of 5.0 in the absence of divalent cations making it a unique acid DNase. Preceding studies suggest that this acid DNase plays an important role in the degradation of exogenous DNA encountered by phagocytosis. The genes encoding the Drosophila melanogaster (fruit fly) and Burkholderia thailandensis (soil bacteria) DNase II enzymes were cloned in our laboratory for the purpose of obtaining sufficient active recombinant protein for protein structure studies. The Burkholderia gene was cloned into the bacterial-expression vector, pET-15b and pET-25b(+) while the Drosophila gene was inserted into pUAST fly expression vector. In both the bacterial and fly expression systems, the DNaseII genes were constructed to express and appended 6 X histidine peptide tag. These His-tag were used to purify the recombinant protein by standard affinity methods. The bacterial system offered the advantage of high-level protein production via induction with IPTG and a variety of E. coli strains while the insect system produced His-tagged proteins that closely resemble the native eukaryotic enzymes. The overexpression of DNaseII in fly utilized the Actin-GAL4 and tubulin-GAL4 system that could be regulated via heat-shock or other tissue-specific promoters. After generation of transgenic flies, larvae expressing the recombinant protein were harvested and their extracts were used to purify the recombinant protein. To our knowledge, this is the first study using transgenic flies for the generation of the recombinant protein for structure studies. DNase II genes from Burkholderia thailandensis and Drosophila melanogaster were successfully cloned and expressed. In the case of Burkholderia thailandensis, active enzyme was obtained after partial purification of the protein in a simple step. In the case of the transgenic fly, our in vivo model, protein was successfully expressed and the expressed protein showed endonuclease activity after partial purification. ^

Subject Area

Biology, Molecular|Biology, Cell

Recommended Citation

Mejia Lara, Adrian Alberto, "Generation and isolation of recombinant DNase II enzyme" (2007). ETD Collection for University of Texas, El Paso. AAI1449725.
http://digitalcommons.utep.edu/dissertations/AAI1449725

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