Cargando…

NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections

Dengue is one of the major diseases causing global public health concerns. Despite technological advances in vaccine production against all its serotypes, it is estimated that the dengue virus is responsible for approximately 390 million infections per year. Laboratory diagnosis has been the key poi...

Descripción completa

Detalles Bibliográficos
Autores principales: Xisto, Mariana Fonseca, Prates, John Willians Oliveira, Dias, Ingrid Marques, Dias, Roberto Sousa, da Silva, Cynthia Canedo, de Paula, Sérgio Oliveira
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345099/
https://www.ncbi.nlm.nih.gov/pubmed/32517281
http://dx.doi.org/10.3390/diagnostics10060379
_version_ 1783556101120196608
author Xisto, Mariana Fonseca
Prates, John Willians Oliveira
Dias, Ingrid Marques
Dias, Roberto Sousa
da Silva, Cynthia Canedo
de Paula, Sérgio Oliveira
author_facet Xisto, Mariana Fonseca
Prates, John Willians Oliveira
Dias, Ingrid Marques
Dias, Roberto Sousa
da Silva, Cynthia Canedo
de Paula, Sérgio Oliveira
author_sort Xisto, Mariana Fonseca
collection PubMed
description Dengue is one of the major diseases causing global public health concerns. Despite technological advances in vaccine production against all its serotypes, it is estimated that the dengue virus is responsible for approximately 390 million infections per year. Laboratory diagnosis has been the key point for the correct treatment and prevention of this disease. Currently, the limiting factor in the manufacture of dengue diagnostic kits is the large-scale production of the non-structural 1 (NS1) antigen used in the capture of the antibody present in the infected patients’ serum. In this work, we demonstrate the production of the non-structural 1 protein of dengue virus (DENV) serotypes 1–4 (NS1-DENV1, NS1-DENV2, NS1-DENV3, and NS1-DENV4) in the methylotrophic yeast Pichia pastoris KM71H. Secreted recombinant protein was purified by affinity chromatography and characterized by SDS-PAGE and ELISA. The objectives of this study were achieved, and the results showed that P. pastoris is a good heterologous host and worked well in the production of NS1DENV 1–4 recombinant proteins. Easy to grow and quick to obtain, this yeast secreted ready-to-use proteins, with a final yield estimated at 2.8–4.6 milligrams per liter of culture. We reached 85–91% sensitivity and 91–93% specificity using IgM as a target, and for anti-dengue IgG, 83–87% sensitivity and 81–93% specificity were achieved. In this work, we conclude that the NS1 recombinant proteins are efficiently produced in P. pastoris and have great potential for use in diagnostic kits for dengue virus infections. The transformed yeast obtained can be used for production in industrial-scale bioreactors.
format Online
Article
Text
id pubmed-7345099
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-73450992020-07-09 NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections Xisto, Mariana Fonseca Prates, John Willians Oliveira Dias, Ingrid Marques Dias, Roberto Sousa da Silva, Cynthia Canedo de Paula, Sérgio Oliveira Diagnostics (Basel) Article Dengue is one of the major diseases causing global public health concerns. Despite technological advances in vaccine production against all its serotypes, it is estimated that the dengue virus is responsible for approximately 390 million infections per year. Laboratory diagnosis has been the key point for the correct treatment and prevention of this disease. Currently, the limiting factor in the manufacture of dengue diagnostic kits is the large-scale production of the non-structural 1 (NS1) antigen used in the capture of the antibody present in the infected patients’ serum. In this work, we demonstrate the production of the non-structural 1 protein of dengue virus (DENV) serotypes 1–4 (NS1-DENV1, NS1-DENV2, NS1-DENV3, and NS1-DENV4) in the methylotrophic yeast Pichia pastoris KM71H. Secreted recombinant protein was purified by affinity chromatography and characterized by SDS-PAGE and ELISA. The objectives of this study were achieved, and the results showed that P. pastoris is a good heterologous host and worked well in the production of NS1DENV 1–4 recombinant proteins. Easy to grow and quick to obtain, this yeast secreted ready-to-use proteins, with a final yield estimated at 2.8–4.6 milligrams per liter of culture. We reached 85–91% sensitivity and 91–93% specificity using IgM as a target, and for anti-dengue IgG, 83–87% sensitivity and 81–93% specificity were achieved. In this work, we conclude that the NS1 recombinant proteins are efficiently produced in P. pastoris and have great potential for use in diagnostic kits for dengue virus infections. The transformed yeast obtained can be used for production in industrial-scale bioreactors. MDPI 2020-06-06 /pmc/articles/PMC7345099/ /pubmed/32517281 http://dx.doi.org/10.3390/diagnostics10060379 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Xisto, Mariana Fonseca
Prates, John Willians Oliveira
Dias, Ingrid Marques
Dias, Roberto Sousa
da Silva, Cynthia Canedo
de Paula, Sérgio Oliveira
NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections
title NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections
title_full NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections
title_fullStr NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections
title_full_unstemmed NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections
title_short NS1 Recombinant Proteins Are Efficiently Produced in Pichia pastoris and Have Great Potential for Use in Diagnostic Kits for Dengue Virus Infections
title_sort ns1 recombinant proteins are efficiently produced in pichia pastoris and have great potential for use in diagnostic kits for dengue virus infections
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345099/
https://www.ncbi.nlm.nih.gov/pubmed/32517281
http://dx.doi.org/10.3390/diagnostics10060379
work_keys_str_mv AT xistomarianafonseca ns1recombinantproteinsareefficientlyproducedinpichiapastorisandhavegreatpotentialforuseindiagnostickitsfordenguevirusinfections
AT pratesjohnwilliansoliveira ns1recombinantproteinsareefficientlyproducedinpichiapastorisandhavegreatpotentialforuseindiagnostickitsfordenguevirusinfections
AT diasingridmarques ns1recombinantproteinsareefficientlyproducedinpichiapastorisandhavegreatpotentialforuseindiagnostickitsfordenguevirusinfections
AT diasrobertosousa ns1recombinantproteinsareefficientlyproducedinpichiapastorisandhavegreatpotentialforuseindiagnostickitsfordenguevirusinfections
AT dasilvacynthiacanedo ns1recombinantproteinsareefficientlyproducedinpichiapastorisandhavegreatpotentialforuseindiagnostickitsfordenguevirusinfections
AT depaulasergiooliveira ns1recombinantproteinsareefficientlyproducedinpichiapastorisandhavegreatpotentialforuseindiagnostickitsfordenguevirusinfections