Part:BBa_K2417010
Ecotin Periplasmic Signal Peptide
Ecotin is a small homodimeric, periplasmic protein found in E. coli (Figure 1). It is 16kDa and is highly stable (can tolerate pH 1 and 100˚C for 30 mins), meaning in conjunction with its N-terminus periplasmic secretion peptide it is an effective periplasmic transporter. When fused to the N-terminus of the protein of interest (tested with human proinsulin), Ecotin transports the protein of interest to the periplasm. The periplasm is a favourable compartment for folding of disulphide bonds due to its oxidative environment, and the presence of isomerases that can correct mismatched disulphide bonds. In the cytoplasm, proteins with exposed disulphide bonds are usually sorted into inclusion bodies due to the reducing environment of the cytosol – which complicates extraction processes, due to the requirement of later in vitro oxidative refolding.
Figure 1: Ecotin peptide showing homodimeric structure, however only a monomer of this peptide is required on the N-terminus of a protein of interest in order to transport it to the periplasm of E. coli. Sourced from: Malik, A. 2016, "Protein fusion tags for efficient expression and purification of recombinant proteins in the periplasmic space of E. coli", 3 Biotech, vol. 6, no. 1, pp. 1-7.
Furthermore, the periplasm has reduced proteases, meaning transported proteins are less likely to be degraded.
The Ecotin signal requires both the signal sequence and the mature protein fused to the N-terminus of the protein (Figure 2). Ecotin has been identified as a model protein for the export of proinsulin into the periplasm of E. coli.
Figure 2: Ecotin transporter peptide as it is found at the N-terminus of proteins of interest, in order to transport it to the periplasm. Both the Ecotin monomer and the periplasmic signal peptide are required for transport.
References:
Malik, A., Jenzsch, M., Lübbert, A., Rudolph, R. & Söhling, B. 2007, "Periplasmic production of native human proinsulin as a fusion to E. coli ecotin", Protein Expression and Purification, vol. 55, no. 1, pp. 100-111.
Winter, J., Neubauer, P., Glockshuber, R. & Rudolph, R. 2000, "Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA", Journal of Biotechnology, vol. 84, no. 2, pp. 175-185.
Malik, A. 2016, "Protein fusion tags for efficient expression and purification of recombinant proteins in the periplasmic space of E. coli", 3 Biotech, vol. 6, no. 1, pp. 1-7.
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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