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Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis

BACKGROUND: Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal pha...

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Autores principales: Koko, Innanurdiani, Song, Adelene Ai-Lian, Masarudin, Mas Jaffri, Abdul Rahim, Raha
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882331/
https://www.ncbi.nlm.nih.gov/pubmed/31775775
http://dx.doi.org/10.1186/s12896-019-0575-x
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author Koko, Innanurdiani
Song, Adelene Ai-Lian
Masarudin, Mas Jaffri
Abdul Rahim, Raha
author_facet Koko, Innanurdiani
Song, Adelene Ai-Lian
Masarudin, Mas Jaffri
Abdul Rahim, Raha
author_sort Koko, Innanurdiani
collection PubMed
description BACKGROUND: Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901–1 integrating system. RESULTS: The constructed integration system comprises of a lactococcal promoter (P(nisA) or P(170)), phage attachment site (attP) from bacteriophage TP901–1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP(344) anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P(170) promoter with USP45 signal peptide which gave very low signals in whole cell ELISA. CONCLUSION: The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.
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spelling pubmed-68823312019-12-03 Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis Koko, Innanurdiani Song, Adelene Ai-Lian Masarudin, Mas Jaffri Abdul Rahim, Raha BMC Biotechnol Research Article BACKGROUND: Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901–1 integrating system. RESULTS: The constructed integration system comprises of a lactococcal promoter (P(nisA) or P(170)), phage attachment site (attP) from bacteriophage TP901–1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP(344) anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P(170) promoter with USP45 signal peptide which gave very low signals in whole cell ELISA. CONCLUSION: The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery. BioMed Central 2019-11-27 /pmc/articles/PMC6882331/ /pubmed/31775775 http://dx.doi.org/10.1186/s12896-019-0575-x Text en © The Author(s). 2019 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
Koko, Innanurdiani
Song, Adelene Ai-Lian
Masarudin, Mas Jaffri
Abdul Rahim, Raha
Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis
title Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis
title_full Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis
title_fullStr Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis
title_full_unstemmed Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis
title_short Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis
title_sort engineering integrative vectors based on phage site-specific recombination mechanism for lactococcus lactis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882331/
https://www.ncbi.nlm.nih.gov/pubmed/31775775
http://dx.doi.org/10.1186/s12896-019-0575-x
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