Abstract
Extracellular secretion is a beneficial way to produce recombinant proteins at an industrial scale. Among bacterial secretion systems, the type 1 secretion system (T1SS) in Gram-negative bacteria is particularly attractive due to its simple architecture involving only three proteins and one-step translocation across both inner and outer membranes. However, proteins that fold rapidly within the cell often fail to pass through the narrow T1SS channel tunnel, limiting its industrial application. To address this limitation, we engineered a 10-amino-acid calcium-binding sequence (CBS) that disrupts proximal secondary structures through electrostatic repulsion at low Ca(2+) concentrations, thereby inhibiting premature folding of target proteins in the cell. We demonstrated that CBS-grafted variants of three fast-folding proteins-mRFP1, RNase H1, and monobody-were efficiently secreted by Escherichia coli expressing the Serratia marcescens Lip T1SS as compared to their parental proteins. Remarkably, the CBS-grafted variants were fully active and structurally identical to the intracellularly produced parental proteins when isolated from culture supernatants. Furthermore, the removal of Ca(2+) from CBS did not compromise the structure or function, indicating that the CBS-mediated calcium-dependent folding was irreversible. Our work will expand the utility of T1SS for secreting diverse proteins, paving the way for broader industrial applications.