pBESTBL1181:IL-10

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In parallel and in order to determine the impact of SPExp4 (issued from L. lactis) for heterologous protein secretion in bifidobacteria, we replaced this SP with a new one issued from B. longum: SP of the hypothetical protein BL1181 from B. longum NCC2705 (Schell et al., 2002). Tthe DNA fragment containing SPBL1181 was obtained from this plasmid with EcoRV/NsiI enzymes (Thermo Scientific) and cloned into purified backbone isolated from blunt-ended-AflIII/NsiI-cut pBESTExp4:IL-10 vector.	The resulting plasmid pBESTBL1181:IL-10 (Figure ​(Figure1)1) was established into B. bifidum.

Upstream tips
In parallel and in order to determine the impact of SPExp4 (issued from L. lactis) for heterologous protein secretion in bifidobacteria, we replaced this SP with a new one issued from B. longum: SP of the hypothetical protein BL1181 from B. longum NCC2705 (Schell et al., 2002). Tthe DNA fragment containing SPBL1181 was obtained from this plasmid with EcoRV/NsiI enzymes (Thermo Scientific) and cloned into purified backbone isolated from blunt-ended-AflIII/NsiI-cut pBESTExp4:IL-10 vector.
Protocol tips
The resulting plasmid pBESTBL1181:IL-10 (Figure ​(Figure1)1) was established into B. bifidum.

Publication protocol

For the expression of IL-10 using BEST system, a DNA fragment encoding for IL-10 mature sequence was obtained from a plasmid harboring murine il-10 gene (cassette SPUsp45:IL-10) under the control of PnisA promoter, also known as pLB270 plasmid (Motta et al., 2012; Benbouziane et al., 2013), with NsiI/SpeI enzymes (Thermo Scientific, Courtaboeuf, France) and cloned into pBESTExp4:Nuc vector digested with the same enzymes and replacing the corresponding nuc DNA fragment. The resulting vector pBESTExp4:IL-10 was established into B. bifidum.

In parallel and in order to determine the impact of SPExp4 (issued from L. lactis) for heterologous protein secretion in bifidobacteria, we replaced this SP with a new one issued from B. longum: SP of the hypothetical protein BL1181 from B. longum NCC2705 (Schell et al., 2002). Indeed, after in silico analysis (using SignalP software1) of homologous proteins efficiently secreted by bifidobacteria we put a particular interest in this SP since it was one with the highest score of “cleavage site.” For the cloning: a DNA fragment containing the SP of BL1181 (SPBL1181) was PCR amplified from genomic DNA of B. longum strain (GenBank Accession Number: NC_004307.2) with the following primers: EcoRV-SPBL1181 (5′-GGATATCAAGGAGATATAAAAATGACGAACGTACGTGTGATCAAGCCCGC-3′) for the coding strand and NsiI-SPBL1181 (5′-TGATGCATCTGCCTGGGCAGGCTGTGCCGAGCTGAACG-3′) for the complementary strand. The PCR product (132 pb) was subcloned into a pCR-TOPO kit (Invitrogen, Carlsbad, CA, United States) resulting in pCR-TOPO:SPBL1181 (Table ​(Table1).1). Afterward, the DNA fragment containing SPBL1181 was obtained from this plasmid with EcoRV/NsiI enzymes (Thermo Scientific) and cloned into purified backbone isolated from blunt-ended-AflIII/NsiI-cut pBESTExp4:IL-10 vector. The resulting plasmid pBESTBL1181:IL-10 (Figure ​(Figure1)1) was established into B. bifidum.

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