RESUMO
BACKGROUND: The hemagglutinin molecule of influenza virus is considered as an ideal model to study biological processes as well as the effect of glycosylation on the function of glycoproteins. OBJECTIVES: The large subunit of the influenza virus A/New Caledonia/20/99 (H1N1) hemagglutinin (HA1) was expressed in recombinant Escherichia coli containing the glycosylation system of Campylobacter jejuni. This viral glycoprotein contains glycosylation motifs recognized by prokaryotic and eukaryotic oligosaccharyltransferases. METHODS: In order to express the hemagglutinin large subunit gene, the gene was amplified using reverse transcription polymerase chain reaction (RT-PCR), and it was cloned in pET22b for periplasmic expression. RESULTS: Western blotting and lectin blotting bands confirmed glycosylation of the HA1 in recombinant E. coli. CONCLUSION: Such a successful accomplishment of hemagglutinin expression in recombinant E. coli can be used to construct carbohydrates in hemagglutinin molecules of different strains in order to produce effective antigens for vaccine and rapid diagnostic kits against new emerging viruses.
Assuntos
Escherichia coli/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Processamento de Proteína Pós-Traducional , Campylobacter jejuni/genética , Escherichia coli/genética , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Redes e Vias Metabólicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The transfer of lipid-linked oligosaccharide to asparagine residues of polypeptide chains is catalyzed by oligosaccharyltransferase (OTase). In most eukaryotes, OTase is a hetero-oligomeric complex composed of eight different proteins, in which the STT3 component is believed to be the catalytic subunit. In the parasitic protozoa Leishmania major, four STT3 paralogues, but no homologues to the other OTase components seem to be encoded in the genome. We expressed each of the four L. major STT3 proteins individually in Saccharomyces cerevisiae and found that three of them, LmSTT3A, LmSTT3B, and LmSTT3D, were able to complement a deletion of the yeast STT3 locus. Furthermore, LmSTT3D expression suppressed the lethal phenotype of single and double deletions in genes encoding other essential OTase subunits. LmSTT3 proteins did not incorporate into the yeast OTase complex but formed a homodimeric enzyme, capable of replacing the endogenous, multimeric enzyme of the yeast cell. Therefore, these protozoan OTases resemble the prokaryotic enzymes with respect to their architecture, but they used substrates typical for eukaryotic cells: N-X-S/T sequons in proteins and dolicholpyrophosphate-linked high mannose oligosaccharides.
