Heterologous protein secretion byLactobacillus plantarumusing homologous signal peptides

Abstract

Aims: To test seven selected putative signal peptides from Lactobacillus plantarum WCFS1 in terms of their ability to drive secretion of two model proteins in Lact. plantarum, and to compare the functionality of these signal peptides with that of well‐known heterologous signal peptides (Usp45, M6). Methods and Results: Signal peptide functionality was assessed using a series of modular derivatives of the pSIP vectors for peptide pheromone‐controlled high‐level gene expression in lactobacilli. Several of the constructs with homologous signal peptides yielded similar or higher reporter protein activities than constructs with heterologous signal peptides. Two of the homologous signal peptides (Lp_0373 and Lp_0600) appeared as especially promising candidates for directing secretion, as they were among the best performing with both reporter proteins. Conclusions: We have identified homologous signal peptides for high‐level secretion of heterologous proteins in Lact. plantarum. With the model proteins, some of these performed better than commonly used heterologous signal peptides. Significance and Impact of the Study: The homologous signal peptides tested out, in this study, could be useful in food‐grade systems for secretion of interesting proteins in Lact. plantarum. The constructed modular secretion vectors are easily accessible for rapid signal peptide screening.

The Major Autolysin Acm2 from Lactobacillus plantarum Undergoes Cytoplasmic O-Glycosylation

“…for protein secretion and anchoring (13,26). These vectors were constructed in order to complement the acm2 mutant with either native Acm2 or an N-terminally truncated version of Acm2 for cytoplasmic expression.…”

RecombinantLactobacillus plantaruminduces immune responses to cancer testis antigen NY-ESO-1 and maturation of dendritic cells

“…38 The code "0373" indicates that secretion of NY-ESO-1 is driven by an N-terminal signal peptide (SP) derived from the L. plantarum protein Lp_0373. 39 The cell wall anchor referred to as cwa3 originates from L. plantarum protein Lp_2578 and comprises 128 residues (see ref. Twenty-three for more details).…”

Cell Wall Anchoring of the 37-Kilodalton Oncofetal Antigen by Lactobacillus plantarum for Mucosal Cancer Vaccine Delivery

“…In this study, we express, secrete, and anchor OFA in the probiotic human saliva isolate Lactobacillus plantarum WCFS1 (14), using the pSIP system (22) for protein expression. All constructed OFA plasmids used in this study are based on pSIP derivatives previously constructed for secretion of staphylococcal nuclease (Nuc) and lactobacillal amylase (Amy) by using a variety of signal peptides (SPs) from L. plantarum WCFS1, which are designated by their gene codes (18,19). Constructs were first established in Escherichia coli TOP10 cells and then transformed into L. plantarum by electroporation, as described previously (3) using the appropriate antibiotics as selection markers.…”

“…AAD26866.1) was amplified from cDNA with primers OFASal (GTCGACTCCGGAGCC CTTGAC) (SalI site italic) and OFAAcc (GGTACCTCAG GACCACTCAGTGGT) (Acc65I site italic). The 0.89-kb OFA fragment was ligated into the SalI-Acc65I-digested vectors pLp_0297sAmyA, pLp_0373sNuc, pLp_0600AmyA, pLp_1447sAmyA, and pLp_2940sAmyA (19), yielding the plasmids pLp_0297sOFA, pLp_0373sOFA, pLp_0600sOFA, pLp_1447sOFA, and pLp_2940sOFA.…”

“…Western blot analysis of protoplast fractions confirmed that OFA was produced by all five SP constructs and the pCytOFA construct (data not shown). Analysis of the secretion efficiency (19) in which a secretion cassette is translationally fused to the inducible P sppA promoter. All parts of the cassette are easily exchangeable using the introduced linker (L) restriction sites (SalI and MluI), the NdeI site at the translational fusion point, and the downstream multiple cloning site (MCS) containing the Acc65I and HindIII sites.…”