title: Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1).
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.
authors.affiliation: Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
authors.forename: Caio Roberto Soares
authors.initials: CR
authors.lastname: Bragança
authors.forename: Lívia Tavares
authors.initials: LT
authors.lastname: Colombo
authors.forename: Alvaro Soares
authors.initials: AS
authors.lastname: Roberti
authors.forename: Mariana Caroline Tocantins
authors.initials: MC
authors.lastname: Alvim
authors.forename: Silvia Almeida
authors.initials: SA
authors.lastname: Cardoso
authors.forename: Kledna Constancio Portes
authors.initials: KC
authors.lastname: Reis
authors.forename: Sérgio Oliveira
authors.initials: SO
authors.lastname: de Paula
authors.forename: Wendel Batista
authors.initials: WB
authors.lastname: da Silveira
authors.forename: Flavia Maria Lopes
authors.initials: FM
authors.lastname: Passos
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D015151:Immunoblotting; D007716:Kluyveromyces / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D015533:Transcriptional Activation; D014170:Transformation, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C082515:NS1 protein, dengue-1 virus; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins