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Among bacterial secretion systems, the type 1 secretion system (T1SS) in Gram\u2010negative bacteria is particularly attractive due to its simple architecture involving only three proteins and one\u2010step 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\u2010amino\u2010acid calcium\u2010binding sequence (CBS) that disrupts proximal secondary structures through electrostatic repulsion at low Ca<jats:sup>2+<\/jats:sup> concentrations, thereby inhibiting premature folding of target proteins in the cell. We demonstrated that CBS\u2010grafted variants of three fast\u2010folding proteins\u2014mRFP1, RNase H1, and monobody\u2014were efficiently secreted by <jats:italic>Escherichia coli<\/jats:italic> expressing the <jats:italic>Serratia marcescens<\/jats:italic> Lip T1SS as compared to their parental proteins. Remarkably, the CBS\u2010grafted variants were fully active and structurally identical to the intracellularly produced parental proteins when isolated from culture supernatants. Furthermore, the removal of Ca<jats:sup>2+<\/jats:sup> from CBS did not compromise the structure or function, indicating that the CBS\u2010mediated calcium\u2010dependent folding was irreversible. Our work will expand the utility of T1SS for secreting diverse proteins, paving the way for broader industrial applications.<\/jats:p>","DOI":"10.1002\/pro.70165","type":"journal-article","created":{"date-parts":[[2025,5,19]],"date-time":"2025-05-19T09:40:46Z","timestamp":1747647646000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Enhanced secretion through type 1 secretion system by grafting a calcium\u2010binding sequence to modify the folding of cargo proteins"],"prefix":"10.1002","volume":"34","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7534-599X","authenticated-orcid":false,"given":"Ryo","family":"Uehara","sequence":"first","affiliation":[{"name":"Department of Biotechnology College of Life Sciences, Ritsumeikan University  Kusatsu Shiga Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuka","family":"Kamiya","sequence":"additional","affiliation":[{"name":"Graduate School of 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