caa a22 4500 605501815 CHVBK 20210128100602.0 cr unu---uuuuu 210128e20150201xx s 000 0 eng 10.1007/s00253-014-5963-5 doi (NATIONALLICENCE)springer-10.1007/s00253-014-5963-5 Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1) [Elektronische Daten] [Caio Bragança, Lívia Colombo, Alvaro Roberti, Mariana Alvim, Silvia Cardoso, Kledna Reis, Sérgio de Paula, Wendel da Silveira, Flavia Passos] 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 200μgmL−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 96h 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. Springer-Verlag Berlin Heidelberg, 2014 Kluyveromyces marxianus nationallicence Heterologous expression nationallicence Dengue virus nationallicence NS1 protein nationallicence Bragança Caio Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut Colombo Lívia Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut Roberti Alvaro Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut Alvim Mariana Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut Cardoso Silvia Laboratório de Imunovirologia Molecular, Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut Reis Kledna Microbiologia Veterinária Especial - MICROVET, Viçosa, MG, Brazil aut de Paula Sérgio Laboratório de Imunovirologia Molecular, Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut da Silveira Wendel Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut Passos Flavia Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/3(2015-02-01), 1191-1203 0175-7598 99:3<1191 2015 99 253 https://doi.org/10.1007/s00253-014-5963-5 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-5963-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Bragança Caio Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut NATIONALLICENCE 700 1- Colombo Lívia Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut NATIONALLICENCE 700 1- Roberti Alvaro Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut NATIONALLICENCE 700 1- Alvim Mariana Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut NATIONALLICENCE 700 1- Cardoso Silvia Laboratório de Imunovirologia Molecular, Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut NATIONALLICENCE 700 1- Reis Kledna Microbiologia Veterinária Especial - MICROVET, Viçosa, MG, Brazil aut NATIONALLICENCE 700 1- de Paula Sérgio Laboratório de Imunovirologia Molecular, Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut NATIONALLICENCE 700 1- da Silveira Wendel Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut NATIONALLICENCE 700 1- Passos Flavia Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/3(2015-02-01), 1191-1203 0175-7598 99:3<1191 2015 99 253