Part:BBa_K2417011
YncM Bacillus Secretion Peptide
Bacillus subtilis is a commonly used organism for the production and secretion of recombinant proteins. Unlike gram positive bacteria, Bacillus can be induced to secrete protein products directly into the surrounding media, eliminating many steps of the protein purification process, and removes the issue of inclusion body formation. In order to secrete a protein, the recombinant protein must have a secretion tag at its N-terminus. YncM is one such tag, belonging to the Sec-type family, and has been shown to have high secretion efficiency.
Bacillus is also a useful vector because its lack of lipopolysaccharide (LPS) endotoxin removes the need to remove it from the media. Similar to the secretion of recombinant proteins to the periplasm, secretion to the media removes proteins from the reducing environment of the cytoplasm, allowing proteins with disulphide bonds to fold more efficiently.
Characterisation
We showed that YncM was an efficient transporter of proteins out of the cell, by fusing it to the N-terminus of an insulin analogue (Winsulin – part BBa_K2417007) and proving it was present in the surrounding media and not the whole cell lysate (Figure 1).
Figure 1: ELISA assay confirms correct folding and structure of proinsulin and Winsulin constructs. ELISA assay was performed on cell lysates for all constructs (additionally, we tested the surrounding media for YncM Winsulin), and showed the presence of Proinsulin or Winsulin in cells expressing Cytoplasmic Proinsulin, Cytoplasmic Winsulin, Ecotin Proinsulin and YncM Winsulin. The assay also proved their ability to bind anti-insulin antibodies, thus suggesting proper folding and structure. 5µL of cell lysates were tested at multiple dilutions, shown here are the 1:1 dilutions. The inclusion of a whole-cell lysate control for YncM Winsulin proves the YncM secretion tag was effectively causing Bacillus secretion of Winsulin into the surrounding media, as it was not found in the cell lysate fraction.
References:
Guan, C., Cui, W., Cheng, J., Liu, Z., Liu, R., Zhou, L. & Zhou, Z. 2016, "Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis", New BIOTECHNOLOGY, vol. 33, no. 3, pp. 372-379.
Westers, L., Westers, H. & Quax, W. 2004, "Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism", BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, vol. 1694, no. 1-3, pp. 299-310.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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