Enhanced secretion through type 1 secretion system by grafting a calcium‐binding sequence to modify the folding of cargo proteins DOI
Ryo Uehara, Yuji Kamiya,

S Maeda

и другие.

Protein Science, Год журнала: 2025, Номер 34(6)

Опубликована: Май 19, 2025

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

Язык: Английский

Enhanced secretion through type 1 secretion system by grafting a calcium‐binding sequence to modify the folding of cargo proteins DOI
Ryo Uehara, Yuji Kamiya,

S Maeda

и другие.

Protein Science, Год журнала: 2025, Номер 34(6)

Опубликована: Май 19, 2025

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

Язык: Английский

Процитировано

0