Cloning and expression of Aedes aegypti heme degradation enzyme
Brewer, L.L; Oliveira, PL and Paiva-Silva, GO
Instituto de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro, Brasil
Aedes aegypti is the vector of Dengue and Yellow Fever, two important human diseases. Its females, as most of hematophagous insects, ingest large amounts of blood in a single meal. Blood digestion releases large amounts of free heme in the insect midgut lumen. Free heme generates powerful reactive oxygen species, which damage a lot of biological components of the cells, like lipids, proteins and DNA. Therefore, this insect has developed several mechanisms to deal with this oxidative challenge, which allowed it to adopt blood as source of nutrients. We have shown that one of these mechanisms is the enzymatic degradation of heme catalised by Heme Oxygenase (HO) that produces biliverdin IX, Fe+2 and CO.
In this work, we isolated Heme Oxygenase gene from midgut of blood-fed females by RT-PCR using specific primers. The full-length sequence of HO cDNA encodes a protein with high similarity with other animals HO, including the insects Drosophila melanogaster and Anopheles gambiae. For protein expression we produced the full-length cDNA and a truncated cDNA without the 21 amino acids of COOH-terminal, responsible for the insertion of HO in the microsomal membranes.
HO cDNA full-length and truncated forms were cloned in pDONR 201 vector followed by recombination into the expression vector pDEST 17, using Gateway cloning system. DH10B and BL21 E. coli strains were transformed for stocking and protein expression respectively. Finally, we intend to purify the recombinant proteins to crystallize them and determine their structure – free and bound to heme – by X-Ray diffraction.
Supported by CNPq, FAPERJ, PRONEX, PADCT and HHMI.
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