Cargando…

Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production

BACKGROUND: The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous repo...

Descripción completa

Detalles Bibliográficos
Autores principales: Zhang, Xiaoyue, Xu, Keyan, Ou, Yanmei, Xu, Xiaodong, Chen, Hongying
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930690/
https://www.ncbi.nlm.nih.gov/pubmed/29720159
http://dx.doi.org/10.1186/s12896-018-0434-1
_version_ 1783319522185314304
author Zhang, Xiaoyue
Xu, Keyan
Ou, Yanmei
Xu, Xiaodong
Chen, Hongying
author_facet Zhang, Xiaoyue
Xu, Keyan
Ou, Yanmei
Xu, Xiaodong
Chen, Hongying
author_sort Zhang, Xiaoyue
collection PubMed
description BACKGROUND: The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. RESULTS: In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. CONCLUSIONS: The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.
format Online
Article
Text
id pubmed-5930690
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-59306902018-05-09 Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production Zhang, Xiaoyue Xu, Keyan Ou, Yanmei Xu, Xiaodong Chen, Hongying BMC Biotechnol Research Article BACKGROUND: The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. RESULTS: In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. CONCLUSIONS: The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV. BioMed Central 2018-05-02 /pmc/articles/PMC5930690/ /pubmed/29720159 http://dx.doi.org/10.1186/s12896-018-0434-1 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Zhang, Xiaoyue
Xu, Keyan
Ou, Yanmei
Xu, Xiaodong
Chen, Hongying
Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production
title Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production
title_full Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production
title_fullStr Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production
title_full_unstemmed Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production
title_short Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production
title_sort development of a baculovirus vector carrying a small hairpin rna for suppression of sf-caspase-1 expression and improvement of recombinant protein production
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930690/
https://www.ncbi.nlm.nih.gov/pubmed/29720159
http://dx.doi.org/10.1186/s12896-018-0434-1
work_keys_str_mv AT zhangxiaoyue developmentofabaculovirusvectorcarryingasmallhairpinrnaforsuppressionofsfcaspase1expressionandimprovementofrecombinantproteinproduction
AT xukeyan developmentofabaculovirusvectorcarryingasmallhairpinrnaforsuppressionofsfcaspase1expressionandimprovementofrecombinantproteinproduction
AT ouyanmei developmentofabaculovirusvectorcarryingasmallhairpinrnaforsuppressionofsfcaspase1expressionandimprovementofrecombinantproteinproduction
AT xuxiaodong developmentofabaculovirusvectorcarryingasmallhairpinrnaforsuppressionofsfcaspase1expressionandimprovementofrecombinantproteinproduction
AT chenhongying developmentofabaculovirusvectorcarryingasmallhairpinrnaforsuppressionofsfcaspase1expressionandimprovementofrecombinantproteinproduction