Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.

Human cytomegalovirus congenital infection represents an unmet medical issue and attempts are ongoing to develop an effective vaccine. The virion fusion players of this enveloped virus are the natural targets to achieve this goal and to develop novel anti-viral therapies. The secreted ectodomain of...

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Autores principales:	Marco Patrone, Nuno Carinhas, Marcos Q Sousa, Cristina Peixoto, Claudio Ciferri, Andrea Carfì, Paula M Alves
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/641976275f174f1a8cd3ade00a6bfc6e
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spelling	oai:doaj.org-article:641976275f174f1a8cd3ade00a6bfc6e2021-11-18T08:29:47ZEnhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.1932-620310.1371/journal.pone.0090753https://doaj.org/article/641976275f174f1a8cd3ade00a6bfc6e2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24595278/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Human cytomegalovirus congenital infection represents an unmet medical issue and attempts are ongoing to develop an effective vaccine. The virion fusion players of this enveloped virus are the natural targets to achieve this goal and to develop novel anti-viral therapies. The secreted ectodomain of the viral fusion factor glycoprotein B (gB) has been exploited so far as an alternative to the cumbersome expression of the wild type trans-membrane protein. In the soluble form, gB showed encouraging but limited potential as antigen candidate calling for further efforts. Here, the exhaustive evaluation of the Baculovirus/insect cell expression system has been coupled to an orthogonal screening for expression additives to produce full-length gB. In detail, rapamycin was found to prolong gB intracellular accumulation while inhibiting the infection-induced cell swelling. Not obvious to predict, this inhibition did not affect Baculovirus growth, revealing that the virus-induced cell size increase is a dispensable side phenotype. In parallel, a feeding strategy for the limiting nutrient cysteine has been set up which improved gB stability. This multi-modal scheme allowed the production of full-length, mutation-free gB in the milligram scale. The recombinant full-length gB obtained was embedded into a stable mono-dispersed particle substantially larger than the protein trimer itself, according to the reported association of this protein with detergent-resistant lipid domains.Marco PatroneNuno CarinhasMarcos Q SousaCristina PeixotoClaudio CiferriAndrea CarfìPaula M AlvesPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 3, p e90753 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Marco Patrone Nuno Carinhas Marcos Q Sousa Cristina Peixoto Claudio Ciferri Andrea Carfì Paula M Alves Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.
description	Human cytomegalovirus congenital infection represents an unmet medical issue and attempts are ongoing to develop an effective vaccine. The virion fusion players of this enveloped virus are the natural targets to achieve this goal and to develop novel anti-viral therapies. The secreted ectodomain of the viral fusion factor glycoprotein B (gB) has been exploited so far as an alternative to the cumbersome expression of the wild type trans-membrane protein. In the soluble form, gB showed encouraging but limited potential as antigen candidate calling for further efforts. Here, the exhaustive evaluation of the Baculovirus/insect cell expression system has been coupled to an orthogonal screening for expression additives to produce full-length gB. In detail, rapamycin was found to prolong gB intracellular accumulation while inhibiting the infection-induced cell swelling. Not obvious to predict, this inhibition did not affect Baculovirus growth, revealing that the virus-induced cell size increase is a dispensable side phenotype. In parallel, a feeding strategy for the limiting nutrient cysteine has been set up which improved gB stability. This multi-modal scheme allowed the production of full-length, mutation-free gB in the milligram scale. The recombinant full-length gB obtained was embedded into a stable mono-dispersed particle substantially larger than the protein trimer itself, according to the reported association of this protein with detergent-resistant lipid domains.
format	article
author	Marco Patrone Nuno Carinhas Marcos Q Sousa Cristina Peixoto Claudio Ciferri Andrea Carfì Paula M Alves
author_facet	Marco Patrone Nuno Carinhas Marcos Q Sousa Cristina Peixoto Claudio Ciferri Andrea Carfì Paula M Alves
author_sort	Marco Patrone
title	Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.
title_short	Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.
title_full	Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.
title_fullStr	Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.
title_full_unstemmed	Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.
title_sort	enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/641976275f174f1a8cd3ade00a6bfc6e
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Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.

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