IndraLab

"To overcome this inconvenience, we then constructed a novel recombinant strain, L. lactis MG1363 harboring an expression plasmid pSECN, in which the n gene was fused with the coding sequence of USP45 signal peptide and was controlled by the constitutive L. lactis promoter P59."

"Our data confirmed that the use of sp6 anchor in addition to usp45 signal peptide and interval sequence can efficiently display LTB on the surface of L. lactis."

"shuttle vector: 1) L. lactis expressing secreted proteins was constructed with recombinant pTRKH2 (secreted-protein plasmid) carrying the Usp45 signal peptide (SPUsp45), nontoxic adjuvanted tetanus toxin fragment C (TETC), and 14 of the 38 C-terminal repeats (14CDTA) of nontoxic C. difficile toxin A (TcdA); and 2) L. lactis expressing secreted and membrane proteins was constructed with recombinant pTRKH2 (membrane-anchored plasmid) carrying SPUsp45, TETC, 14CDTA, and the cell wall-anchored sequence of protein M6 (cwaM6)."

"P1 represents the constitutive promoter; SD, Shine-Dalgarno sequence; SS, first codons of the Usp45 signal peptide fused to the psaA gene."

"P1 represents the constitutive promoter; SD, Shine-Dalgarno sequence; SS, first codons of the Usp45 signal peptide fused to the psaA gene.Fig."

"The psaA gene was cloned into the pT1NX vector PstI site, which produced a fusion of a truncated usp45 signal peptide carried by the vector to the PsaA sequence (Fig. 1), under the control of a constitutive promoter."

"To calculate the efficacy of hsTRAIL secretion, when pNZ8124 plasmid vector with sequence for usp45 leader peptide was used, we performed ELISA and quantified the concentration of the protein both in crude supernatants and cell lysates after 4 h upon the nisin-induction."

"In early work in the field, Dieye et al. [27] showed that replacement of the M6 signal peptide with the Usp45 signal peptide from L. lactis doubled the amount of a staphylococcal nuclease reporter protein (NucA) that was successfully anchored to the cell wall [27] ."

"A cassette consisting of USP45 fused with LEISSTCDA propeptide to increase secretion efficiency, pNZ8048 multiple cloning sites (MCS) modified to include more RE sites, pNZ8048 terminator (T), and TP901–1 bacteriophage attachment site (attP) was synthesized by Integrated DNA Technologies (Singapore) and denoted as attP cassette.The vectors constructions strategy was divided into four phases; Phase I) Construction of PZatt cassette composed of the P promoter, USP45 signal peptide, LEISSTCDA propeptide and attP sequence; Phase II) Construction of the secretion integrative vectors (pS1–4); (a) Plasmid circularisation by incorporation of erythromycin resistant gene and E. coli replication origin into the PZatt cassette, denoted as pS1; (b) Replacement of P promoter with P promoter to produce pS3 plasmid; (c) Replacement of USP45-LEISSTCDA fusion with SPK1 signal peptide into each pS1 and pS3 plasmids to produce pS2 and pS4 plasmids (Additional file 3: Figure S1); Phase III) Construction of the surface display integrative vectors (pSD1–4) by incorporation of PrtP anchor domain into each secretion integrative vector; Phase IV) Cloning of reporter gene, nuclease (Additional file 4: Figure S2)."

"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."

"Whole cell ELISA assay showed that pSD1nuc which had the combination of P promoter with USP45 signal peptide gave very low signals in the ELISA assay, although fluorescent signals were still detectable in immunofluorescence assay (Fig. 7)."

"For anchoring EG95 protein to the cell surface, we utilized Usp45 signal peptide at the N-terminal and the M6 cell-wall anchor at the C-terminal of the protein, and our results showed that EG95 could effectively be exhibited at the cell wall of L. lactis (Fig. 4)."

"On the other hand, the pS2 plasmid was constructed by replacing USP45 signal peptide with SPK1 using EcoRV and KpnI RE."

"A new VP1 (SPVP1), which was codon optimized with L. lactis, was synthesized by Nanjing GenScript Co. Ltd and then incorporated into pUC57 at the NcoI restriction site and contained a secretory Usp45 signal peptide (SPusp45) at the N terminus of the VP1 gene and the HindIII restriction site at the C terminus."

"Substantially higher levels of the fusion proteins (four- to six-fold) were secreted by the clones possessing Lactobacillus brevis SlpA signal peptide than by those possessing L. lactis Usp45 signal peptide."

"The subsequent amino acid sequence-mass spectrometry sequencing of protein content in the upper and bottom TRAIL bands, revealed that in both bands TRAIL protein occurs beyond any doubt (Table 1) and in both TRAIL fractions the usp45 leader peptide (27 aa) was cut off."

"P values < 0.05 were considered to be significant.The food-grade expression vector pNZ-SECF1S1 harboring the nisin-inducible promoter (PnisA) and Usp45 signal peptide was constructed."