9LSF

Crystal structure of mRFP1 with a grafted calcium-binding sequence and one bound calcium ion in a calcium-free solution

9LSF の概要

• エントリーDOI: 10.2210/pdb9lsf/pdb
• 分子名称: Red fluorescent protein,grafted calcium-binding sequence, CALCIUM ION, DI(HYDROXYETHYL)ETHER, ... (5 entities in total)
• 機能のキーワード: protein engineering, fluorescent protein, mrfp1, calcium-binding sequence
• 由来する生物種: Discosoma; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 63110.08

構造登録者

Uehara, R.,Kamiya, Y.,Maeda, S.,Okamoto, K.,Toya, S.,Chiba, R.,Amesaka, H.,Takano, K.,Matsumura, H.,Tanaka, S.-i. (登録日: 2025-02-04, 公開日: 2025-06-04, 最終更新日: 2026-03-18)

主引用文献

Uehara, R.,Kamiya, Y.,Maeda, S.,Okamoto, K.,Toya, S.,Chiba, R.,Amesaka, H.,Takano, K.,Matsumura, H.,Tanaka, S.I.
Enhanced secretion through type 1 secretion system by grafting a calcium-binding sequence to modify the folding of cargo proteins.
Protein Sci., 34:e70165-e70165, 2025

PubMed 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 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 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.

PubMed: 40384617
DOI: 10.1002/pro.70165

実験手法

X-RAY DIFFRACTION (1.62 Å)