Cloning, Expression and Functional Characterization of Dengue Virus 2 Glycoprotein E Expressed in Pichia pastoris
Allonso, D.1; Pereira, I.B.2; Freitas-Correa, L.1; Kurtenbach, E.2; Da Poian, A.T.2; Mohana-Borges, R.1
1- Laboratório de Genômica Estrutural, IBCCF, UFRJ; 2- Instituto de Bioquímica Médica, IBqM, UFRJ
Dengue virus is a member of Flaviviridae family and represents one of the most important mosquito-borne pathogens. It causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Since the last decade, this virus has been a major threat for the society worldwide. Dengue virus is 50nm-diameter enveloped virus that contains a single-strand and positive-polarity RNA of about 10.7kb. This genome encodes three structural proteins (envelope glycoprotein, E, capsid, C and membrane, M) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). E glycoprotein is the major protein of the envelope exposed on the surface of the particle as homodimers. Each monomer consists of three domains; domain I is responsible for structure of the protein; domain II is the dimerization domain that contains the fusion peptide, essential for virus-cell fusion; and domain III is believed to interact with putative cellular receptors.
Our aim is to characterize functionally the E glycoprotein during the fusion process, to identify the cell receptor responsible for endocytosis of the virus, and to try to discover an inhibitor of this process. Although transformation into bacteria is simpler and more straightforward, we did not have success in expressing the E glycoprotein in this type of cell. Therefore, we decided to express it in Pichia pastoris system. First, we inserted a 6xHistidine tag and an nTev protease cleavage site after the a-signal peptide sequence present in the pPICZα B plasmid in order to facilitate the protein purification steps. Next, to this modified vector (called pPICZaB-6xHisTag-Tev), we cloned the E glycoprotein gene obtained from a plasmid containing the whole dengue virus 2 genome by PCR amplification. The new chimera vector was linearized by Sac I enzyme and transformed into Pichia pastoris yeast cells (strain SMD 1168). The E glycoprotein containing clones were selected by zeocin antibiotic. The protein expression was carried out in BMMY and the highest expression level was obtained after 96 hours with a daily methanol induction. The protein expression was also detected by Western Blotting using a 6xHis-tag antibody. Currently, we are carrying out the fusion experiments with the purified E glycoprotein.
Funded by: CNPq, WHO/TDR, FAPERJ, PRONEX-RIO, IMBEB2.
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