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...
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis |
| Language: | English English |
| Published: |
2021
|
| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/112997/1/112997.pdf |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | 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. |
|---|