Modification of signal peptide for enhanced secretion in Lactoccoccus lactis for oral vaccine delivery

Recombinant heterologous protein secretion is essential in biomanufacturing. Successful protein secretion generally depends on the host strain, expression and secretion machinery, and the target protein. Lactococcus lactis, which has a long history of safe use in food production, has been the wor...

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Main Author:	Alias, Nur Aqlili Riana
Format:	Thesis
Language:	English English
Published:	2021
Subjects:	Lactococcus lactis Oral vaccines
Online Access:	http://psasir.upm.edu.my/id/eprint/112997/1/112997.pdf
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id	my-upm-ir.112997
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spelling	my-upm-ir.1129972024-10-23T08:24:24Z Modification of signal peptide for enhanced secretion in Lactoccoccus lactis for oral vaccine delivery 2021-06 Alias, Nur Aqlili Riana Recombinant heterologous protein secretion is essential in biomanufacturing. Successful protein secretion generally depends on the host strain, expression and secretion machinery, and the target protein. Lactococcus lactis, which has a long history of safe use in food production, has been the workhorse for the secretion of various recombinant heterologous proteins, owing to its monolayer cell wall and presence of only one extracellular housekeeping protease with very few native extracellular proteins for simple and cost-efficient product recovery. Nonetheless, protein secretion in prokaryotes such as L. lactis is often plagued by several limitations such as incomplete translocation, protein misfolding, and degradation, leading to low secretion efficiency. In this study, the main aim is to enhance the low secretion efficiency (SE) in L. lactis by optimization of the secretion system utilizing a novel heterologous signal peptide (SP) SPK1 of Pediococcus pentosaceus. SPK1, which was previously shown to aid comparably, if not better secretion of heterologous proteins than the most widely used lactococcal signal peptide, USP45 was subjected to site- directed mutagenesis (SDM) of its amino acid sequence targeting the tripartite N-, H-, and C- terminal domain, respectively. The effect of SDM on SE was primarily tested on a model protein, Staphylococcus aureus nuclease (NUC). In silico analysis performed on the SPK1 yielded eight putative SPK1 variants; the cassettes of different SPs fused to NUC were cloned in nisin-induced pNZ8048 expression plasmid and introduced into L. lactis NZ9000 host. Analysis of secretion efficiency via Fluorescence Resonance Energy Transfer (FRET) activity assay revealed four of eight SPK1 variants carrying C-domain mutations had successfully enhanced SE compared to both control SPs, SPK1, and native lactococcal USP45. Additionally, one SPK1 variant (SPKM19) showed improved SE by approximately 88% or 1.3-fold than the wild-type SPK1. A subsequent fusion of the SPKM19 with a synthetic propeptide, LEISSTCDA, had further increased the SE. Subsequently, the efficiencies of the SPKM19-LEISS and SPK1-LEISS were further tested on two different therapeutic peptides; a modified 68-V (a derivative of G12V mutant KRAS) fused to carrier molecule diphtheria toxoid (68-V-DT) and a wild-type KRAS (wtKRAS). The SE of the secreted KRAS peptides was determined in vitro and in vivo via oral immunization with mucoadhesive and enteric-coated L. lactis-secreting KRAS in BALB/c mice. Postimmunization assessments on the recombinant L. lactis secreting-KRAS aided by the optimized SPKM19-LEISS revealed a significant elevation in KRAS-specific intestinal IgA titer, indicative of positive induction of humoral immunity. Additionally, despite the lower immune responses observed for SPKM19-treated groups compared to the original SPK1-treated groups, which was consistent with the in vitro findings, secretion of the fusion peptide aided by both SPs to the targeted mucosal site was successfully shown. Altogether, this study demonstrated the development of an enhanced secretory system in L. lactis NZ9000 through an optimized signal peptide SPK1 (SPKM19) and LEISSTCDA, for heterologous protein production and oral vaccine delivery applications. Apart from that, this study also discussed the potential bottlenecks in developing the lactococcal GRAS (Generally-Regarded as Safe) as a secretory host for oral vaccine delivery targeting the mucosal environment. Lactococcus lactis Oral vaccines 2021-06 Thesis http://psasir.upm.edu.my/id/eprint/112997/ http://psasir.upm.edu.my/id/eprint/112997/1/112997.pdf text en public doctoral Universiti Putra Malaysia Lactococcus lactis Oral vaccines Abdul Rahim, Wira Raha English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
advisor	Abdul Rahim, Wira Raha
topic	Lactococcus lactis Oral vaccines
spellingShingle	Lactococcus lactis Oral vaccines Alias, Nur Aqlili Riana Modification of signal peptide for enhanced secretion in Lactoccoccus lactis for oral vaccine delivery
description	Recombinant heterologous protein secretion is essential in biomanufacturing. Successful protein secretion generally depends on the host strain, expression and secretion machinery, and the target protein. Lactococcus lactis, which has a long history of safe use in food production, has been the workhorse for the secretion of various recombinant heterologous proteins, owing to its monolayer cell wall and presence of only one extracellular housekeeping protease with very few native extracellular proteins for simple and cost-efficient product recovery. Nonetheless, protein secretion in prokaryotes such as L. lactis is often plagued by several limitations such as incomplete translocation, protein misfolding, and degradation, leading to low secretion efficiency. In this study, the main aim is to enhance the low secretion efficiency (SE) in L. lactis by optimization of the secretion system utilizing a novel heterologous signal peptide (SP) SPK1 of Pediococcus pentosaceus. SPK1, which was previously shown to aid comparably, if not better secretion of heterologous proteins than the most widely used lactococcal signal peptide, USP45 was subjected to site- directed mutagenesis (SDM) of its amino acid sequence targeting the tripartite N-, H-, and C- terminal domain, respectively. The effect of SDM on SE was primarily tested on a model protein, Staphylococcus aureus nuclease (NUC). In silico analysis performed on the SPK1 yielded eight putative SPK1 variants; the cassettes of different SPs fused to NUC were cloned in nisin-induced pNZ8048 expression plasmid and introduced into L. lactis NZ9000 host. Analysis of secretion efficiency via Fluorescence Resonance Energy Transfer (FRET) activity assay revealed four of eight SPK1 variants carrying C-domain mutations had successfully enhanced SE compared to both control SPs, SPK1, and native lactococcal USP45. Additionally, one SPK1 variant (SPKM19) showed improved SE by approximately 88% or 1.3-fold than the wild-type SPK1. A subsequent fusion of the SPKM19 with a synthetic propeptide, LEISSTCDA, had further increased the SE. Subsequently, the efficiencies of the SPKM19-LEISS and SPK1-LEISS were further tested on two different therapeutic peptides; a modified 68-V (a derivative of G12V mutant KRAS) fused to carrier molecule diphtheria toxoid (68-V-DT) and a wild-type KRAS (wtKRAS). The SE of the secreted KRAS peptides was determined in vitro and in vivo via oral immunization with mucoadhesive and enteric-coated L. lactis-secreting KRAS in BALB/c mice. Postimmunization assessments on the recombinant L. lactis secreting-KRAS aided by the optimized SPKM19-LEISS revealed a significant elevation in KRAS-specific intestinal IgA titer, indicative of positive induction of humoral immunity. Additionally, despite the lower immune responses observed for SPKM19-treated groups compared to the original SPK1-treated groups, which was consistent with the in vitro findings, secretion of the fusion peptide aided by both SPs to the targeted mucosal site was successfully shown. Altogether, this study demonstrated the development of an enhanced secretory system in L. lactis NZ9000 through an optimized signal peptide SPK1 (SPKM19) and LEISSTCDA, for heterologous protein production and oral vaccine delivery applications. Apart from that, this study also discussed the potential bottlenecks in developing the lactococcal GRAS (Generally-Regarded as Safe) as a secretory host for oral vaccine delivery targeting the mucosal environment.
format	Thesis
qualification_level	Doctorate
author	Alias, Nur Aqlili Riana
author_facet	Alias, Nur Aqlili Riana
author_sort	Alias, Nur Aqlili Riana
title	Modification of signal peptide for enhanced secretion in Lactoccoccus lactis for oral vaccine delivery
title_short	Modification of signal peptide for enhanced secretion in Lactoccoccus lactis for oral vaccine delivery
title_full	Modification of signal peptide for enhanced secretion in Lactoccoccus lactis for oral vaccine delivery
title_fullStr	Modification of signal peptide for enhanced secretion in Lactoccoccus lactis for oral vaccine delivery
title_full_unstemmed	Modification of signal peptide for enhanced secretion in Lactoccoccus lactis for oral vaccine delivery
title_sort	modification of signal peptide for enhanced secretion in lactoccoccus lactis for oral vaccine delivery
granting_institution	Universiti Putra Malaysia
publishDate	2021
url	http://psasir.upm.edu.my/id/eprint/112997/1/112997.pdf
_version_	1818586131303759872

Modification of signal peptide for enhanced secretion in Lactoccoccus lactis for oral vaccine delivery

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