Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1)

• Autor(a) principal: Bragança, Caio Roberto Soares
• Data de Publicação: 2010
• Outros Autores: 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
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: https://doi.org/10.1007/s00253-014-5963-5; http://www.locus.ufv.br/handle/123456789/18300
• topic: Kluyveromyces marxianus; Heterologous expression; Dengue virus; NS1 protein

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.