RESUMO
Thioaptamers targeting the dengue-2 virus (DENV-2) envelope protein domain III (EDIII) were developed. EDIII, which contains epitopes for binding neutralizing antibodies, is the putative host-receptor binding domain and is thus an attractive target for development of vaccines, anti-viral therapeutic and diagnostic agents. Thioaptamer DENTA-1 bound to DENV-2 EDIII adjacent to a known neutralizing antibody binding site with a dissociation constant of 154nM.
Assuntos
Antivirais/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Vírus da Dengue/efeitos dos fármacos , Proteínas do Envelope Viral/efeitos dos fármacos , Anticorpos Neutralizantes/imunologia , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Vírus da Dengue/química , Espectroscopia de Ressonância Magnética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologiaRESUMO
Inclusion bodies (IBs) are commonly formed in Escherichia coli due to over expression of recombinant proteins in non-native state. Isolation, denaturation and refolding of these IBs is generally performed to obtain functional protein. However, during this process IBs tend to form non-specific interactions with sheared nucleic acids from the genome, thus getting carried over into downstream processes. This may hinder the refolding of IBs into their native state. To circumvent this, we demonstrate a methodology termed soni-removal which involves disruption of nucleic acid-inclusion body interaction using sonication; followed by solvent based separation. As opposed to conventional techniques that use enzymes and column-based separations, soni-removal is a cost effective alternative for complete elimination of buried and/or strongly bound short nucleic acid contaminants from IBs.