Coding
SP1-NSP4

Part:BBa_K2760000:Design

Designed by: Mariano del Toro   Group: iGEM18_TecMonterrey_GDL   (2018-10-03)

SP1-NSP4: Signal Peptide 1 - Novel Signal Peptide 4

This signal peptide was taken from Han et al 2017 [1], the novel signal peptide 4 (NSP4) was selected because it improved the secretion of a desired target and even decreased induction time. The novel peptides designed in the study can be used to improve the secretion efficiency of other recombinant proteins , for E. coli expression platforms. This signal peptide is part of the Sec-dependent (Sec) secretion pathway, the general secretion E. coli route. Signal peptides linked to the N-terminus of recombinant proteins play a critical role in translocation and secretion [1].

It’s worth noting that both novel signal peptides were designed and derived of certain domains present in the two most efficient E. coli signal peptides studied by Han et al 2017, one from the Sec pathway phoAss and one from the SRP pathway (dsbAss) [1].

Signal peptides are short peptides (20–30 amino acid residues) that have three structural domains; an amino-terminal region with a net positive charge (the n-region), then an hydrophobic region (the h-region) and a protease recognition sequence (the c-region) with a preference for small residues at the −3 (P3) and −1 (P1) positions relative to the cleavage site [1]. Studies show that the hydrophobic region in the signal peptide plays an important role for protein translocation across the bacterial cytoplasmic membrane due to the interaction of the h-region with the membrane during protein translocation. Several studies have also reported that translocation efficiency increases with the length and hydrophobicity of the h-region, and a minimum hydrophobicity is required for their secretion function [1].

Using a molecular weight calculator online the protein weight for the amino acid sequence of the signal peptide resulted in 1.98 kilodaltons [2].

    • The sequence is codon optimized mainly for E.coli however it is meant to be tested in L. rhamnosus as well

References: [1] Han, S., Machhi, S., Berge, M., Xi, G., Linke, T., & Schoner, R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli. AMB Express, 7, 93. http://doi.org/10.1186/s13568-017-0394-1

[2] Molecular weight calculator. Science Gateway. https://www.sciencegateway.org/tools/proteinmw.htm