Engineering integrative vectors based on bacteriophage site specific recombination mechanism for heterologous expression of secreted and surface-anchored protein in Lactococcus lactis
Bacterial integrating system allows integration of foreign DNA into bacterial host chromosome enabling stable expression of foreign gene for recombinant protein production. In this study, integrative expression vectors for secretion and surface display of heterologous protein in Lactococcus lacti...
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my-upm-ir.921402023-11-16T02:39:17Z Engineering integrative vectors based on bacteriophage site specific recombination mechanism for heterologous expression of secreted and surface-anchored protein in Lactococcus lactis 2019-07 Koko, Innanurdiani Bacterial integrating system allows integration of foreign DNA into bacterial host chromosome enabling stable expression of foreign gene for recombinant protein production. In this study, integrative expression vectors for secretion and surface display of heterologous protein in Lactococcus lactis have been successfully constructed based on the site-specific recombination mechanism, of temperate lactococcal phage TP901-1. Two variations of integrative vectors were constructed, denoted pS1-4, and pSD1-4, which allows the heterologous protein to be secreted into the extracellular environment or surface displayed on L. lactis, respectively. The integrative vectors composed of (i) P170 autoinducible promoter or PnisA inducible promoter, (ii) multiple cloning sites (MCS), (iii) TP901-1 bacteriophage attachment site, attP, (iv) signal peptide-encoding sequence USP45 fused with LEISSTCDA propeptide or SPK1 signal peptide for extracellular targeting, and (v) 344 amino acids of proteinase anchor domain (PrtP344) for surface display application. A helper plasmid harbouring int gene, pNZint was also constructed to facilitate plasmid integration into the genome. A staphylococcal nuclease reporter gene was cloned into each of the constructed integrative vectors which were then successfully integrated into the L. lactis genome. Toluidine blue O-DNA assay and immunofluorescence microscopy data proved that the expressed nuclease was able to be secreted or anchored on the L. lactis cell wall. From the findings, signal peptide SPK1 was shown to be superior over USP45 in the secretion of Nuc, even though the USP45 was fused with LEISSTCDA propeptide which reportedly could enhance protein secretion. Meanwhile, the expression of Nuc showed that integrative vectors driven by P170 promoter have comparable strength to PnisA promoter. However, the combination of P170 with USP45-LEISSTCDA fusion performed significantly worse than the other constructs for surface display of Nuc. Therefore, these newly constructed synthetic integrative vectors can be applied for the secretion and surface display of heterologous protein in L. lactis for research or industrial purposes where strong and weak stable expression vectors are required. Lactococcus lactis - Biotechnology Lactococcus lactis Genetic vectors 2019-07 Thesis http://psasir.upm.edu.my/id/eprint/92140/ http://psasir.upm.edu.my/id/eprint/92140/1/FBSB%202019%2029%20UPM%20IR.pdf text en public masters Universiti Putra Malaysia Lactococcus lactis - Biotechnology Lactococcus lactis Genetic vectors Abdul Rahim, Raha |
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Universiti Putra Malaysia |
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PSAS Institutional Repository |
language |
English |
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Abdul Rahim, Raha |
topic |
Lactococcus lactis - Biotechnology Lactococcus lactis Genetic vectors |
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Lactococcus lactis - Biotechnology Lactococcus lactis Genetic vectors Koko, Innanurdiani Engineering integrative vectors based on bacteriophage site specific recombination mechanism for heterologous expression of secreted and surface-anchored protein in Lactococcus lactis |
description |
Bacterial integrating system allows integration of foreign DNA into bacterial
host chromosome enabling stable expression of foreign gene for recombinant
protein production. In this study, integrative expression vectors for secretion
and surface display of heterologous protein in Lactococcus lactis have been
successfully constructed based on the site-specific recombination mechanism,
of temperate lactococcal phage TP901-1. Two variations of integrative vectors
were constructed, denoted pS1-4, and pSD1-4, which allows the heterologous
protein to be secreted into the extracellular environment or surface displayed
on L. lactis, respectively. The integrative vectors composed of (i) P170 autoinducible
promoter or PnisA inducible promoter, (ii) multiple cloning sites (MCS),
(iii) TP901-1 bacteriophage attachment site, attP, (iv) signal peptide-encoding
sequence USP45 fused with LEISSTCDA propeptide or SPK1 signal peptide
for extracellular targeting, and (v) 344 amino acids of proteinase anchor
domain (PrtP344) for surface display application. A helper plasmid harbouring int
gene, pNZint was also constructed to facilitate plasmid integration into the
genome. A staphylococcal nuclease reporter gene was cloned into each of the
constructed integrative vectors which were then successfully integrated into the
L. lactis genome. Toluidine blue O-DNA assay and immunofluorescence
microscopy data proved that the expressed nuclease was able to be secreted
or anchored on the L. lactis cell wall. From the findings, signal peptide SPK1
was shown to be superior over USP45 in the secretion of Nuc, even though the
USP45 was fused with LEISSTCDA propeptide which reportedly could
enhance protein secretion. Meanwhile, the expression of Nuc showed that
integrative vectors driven by P170 promoter have comparable strength to PnisA
promoter. However, the combination of P170 with USP45-LEISSTCDA fusion
performed significantly worse than the other constructs for surface display of
Nuc. Therefore, these newly constructed synthetic integrative vectors can be
applied for the secretion and surface display of heterologous protein in L. lactis for research or industrial purposes where strong and weak stable expression
vectors are required. |
format |
Thesis |
qualification_level |
Master's degree |
author |
Koko, Innanurdiani |
author_facet |
Koko, Innanurdiani |
author_sort |
Koko, Innanurdiani |
title |
Engineering integrative vectors based on bacteriophage site specific recombination mechanism for heterologous expression of secreted and surface-anchored protein in Lactococcus lactis |
title_short |
Engineering integrative vectors based on bacteriophage site specific recombination mechanism for heterologous expression of secreted and surface-anchored protein in Lactococcus lactis |
title_full |
Engineering integrative vectors based on bacteriophage site specific recombination mechanism for heterologous expression of secreted and surface-anchored protein in Lactococcus lactis |
title_fullStr |
Engineering integrative vectors based on bacteriophage site specific recombination mechanism for heterologous expression of secreted and surface-anchored protein in Lactococcus lactis |
title_full_unstemmed |
Engineering integrative vectors based on bacteriophage site specific recombination mechanism for heterologous expression of secreted and surface-anchored protein in Lactococcus lactis |
title_sort |
engineering integrative vectors based on bacteriophage site specific recombination mechanism for heterologous expression of secreted and surface-anchored protein in lactococcus lactis |
granting_institution |
Universiti Putra Malaysia |
publishDate |
2019 |
url |
http://psasir.upm.edu.my/id/eprint/92140/1/FBSB%202019%2029%20UPM%20IR.pdf |
_version_ |
1794018903731470336 |