Use este identificador para citar ou linkar para este item: https://locus.ufv.br//handle/123456789/18300
Tipo: Artigo
Título: Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1)
Autor(es): Bragança, Caio Roberto Soares
Colombo, Lívia Tavares
Roberti, Alvaro Soares
Alvim, Mariana Caroline Tocantins
Cardoso, Silvia Almeida
Reis, Kledna Constancio Portes
Paula, Sérgio Oliveira de
Silveira, Wendel Batista da
Passos, Flavia Maria Lopes
Abstract: The yeast Kluyveromyces marxianus is a convenient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon-optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α-mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast extract peptone dextrose containing 200 μg mL−1 Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 polypeptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post-growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications.
Palavras-chave: Kluyveromyces marxianus
Heterologous expression
Dengue virus
NS1 protein
Editor: Applied Microbiology and Biotechnology
Tipo de Acesso: Springer-Verlag Berlin Heidelberg
URI: https://doi.org/10.1007/s00253-014-5963-5
http://www.locus.ufv.br/handle/123456789/18300
Data do documento: 2-Abr-2010
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