Exploring the N-terminal role of a heterologous protein in secreting out of Escherichia coli

ABSTRACT Escherichia coli is commonly used as a host for the extracellular production of proteins. However, its secretion capacity is often limited to a frustratingly low level compared with other expression hosts, because E. coli has a complex cell envelope with two layers. In previous report, we identified that the catalytic domain of a cellulase (Cel‐CD) from Bacillus subtilis can be secreted into the medium from recombinant E. coli in large quantities without its native signal peptide. In this study, we proved the N‐terminal sequence of the full length Cel‐CD played a crucial role in transportation through both inner and outer membranes. By subcellular location analysis, we verified that the secretion was a two‐step process via the SecB‐dependent pathway through the inner membrane and an unknown pathway through the outer membrane. However, the N‐terminal region of Cel‐CD is polar and hydrophilic, which showed no similarities to other typical signal sequences. Random mutagenesis experiment suggested that the N‐terminal sequence is a compromising result of transportation through inner and outer membranes. This is the first report that a “non‐classical signal peptide” can guide recombinant proteins out of the cells from cytoplasm. Biotechnol. Bioeng. 2016;113: 2561–2567. © 2016 Wiley Periodicals, Inc. Liang and coworkers have proved that the N‐terminal sequence of the full length a catalytic domain of a cellulose (Cel‐CD) mediates the transportation through both inner and outer membranes via the SecB‐dependent pathway and an unknown pathway, respectively. The authors also verified the property of the N‐terminal region is polar and hydrophilic, which showed no similarities to other typical signal sequences, and the transportation through inner and outer membranes in E. coli is a sequence compromising result.