Manipulating subcellular protein localization to enhance target protein accumulation in minicells
File(s)
Published version
Author(s)
Park, Junhyeon
Polizzi, Karen M
Kim, Jongmin
Kim, Juhyun
Type
Journal Article
Abstract
Background
Minicells are chromosome-free derivatives of bacteria formed through irregular cell division. Unlike simplified structures, minicells retain all cellular components of the parent cell except for the chromosome. This feature reduces immunogenic responses, making them advantageous for various biotechnological applications, including chemical production and drug delivery. To effectively utilize minicells, it is essential to ensure the accumulation of target proteins within them, enhancing their efficiency as delivery vehicles.
Results
In this study, we engineered Escherichia coli by deleting the minCD genes, generating minicell-producing strains, and investigated strategies to enhance protein accumulation within the minicells. Comparative proteomic analysis revealed that minicells retain most parent-cell proteins but exhibit an asymmetric proteome distribution, leading to selective protein enrichment. We demonstrated that heterologous proteins, such as GFP and RFP, accumulate more abundantly in minicells than in parent cells, regardless of expression levels. To further enhance this accumulation, we manipulated protein localization by fusing target proteins to polar localization signals. While proteins fused with PtsI and Tsr exhibited 2.6-fold and 2.8-fold increases in accumulation, respectively, fusion with the heterologous PopZ protein resulted in a remarkable 15-fold increase in protein concentration under low induction conditions.
Conclusions
These findings highlight the critical role of spatial protein organization in enhancing the cargo-loading capabilities of minicells. By leveraging polar localization signals, this work provides a robust framework for optimizing minicells as efficient carriers for diverse applications, from therapeutic delivery to industrial biomanufacturing.
Minicells are chromosome-free derivatives of bacteria formed through irregular cell division. Unlike simplified structures, minicells retain all cellular components of the parent cell except for the chromosome. This feature reduces immunogenic responses, making them advantageous for various biotechnological applications, including chemical production and drug delivery. To effectively utilize minicells, it is essential to ensure the accumulation of target proteins within them, enhancing their efficiency as delivery vehicles.
Results
In this study, we engineered Escherichia coli by deleting the minCD genes, generating minicell-producing strains, and investigated strategies to enhance protein accumulation within the minicells. Comparative proteomic analysis revealed that minicells retain most parent-cell proteins but exhibit an asymmetric proteome distribution, leading to selective protein enrichment. We demonstrated that heterologous proteins, such as GFP and RFP, accumulate more abundantly in minicells than in parent cells, regardless of expression levels. To further enhance this accumulation, we manipulated protein localization by fusing target proteins to polar localization signals. While proteins fused with PtsI and Tsr exhibited 2.6-fold and 2.8-fold increases in accumulation, respectively, fusion with the heterologous PopZ protein resulted in a remarkable 15-fold increase in protein concentration under low induction conditions.
Conclusions
These findings highlight the critical role of spatial protein organization in enhancing the cargo-loading capabilities of minicells. By leveraging polar localization signals, this work provides a robust framework for optimizing minicells as efficient carriers for diverse applications, from therapeutic delivery to industrial biomanufacturing.
Date Issued
2025-03-29
Date Acceptance
2025-03-17
Citation
Journal of Biological Engineering, 2025, 19
ISSN
1754-1611
Publisher
Springer Science and Business Media LLC
Journal / Book Title
Journal of Biological Engineering
Volume
19
Copyright Statement
© The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/40158151
PII: 10.1186/s13036-025-00495-y
Subjects
Minicells
Polar localization
PopZ
Publication Status
Published
Coverage Spatial
England
Article Number
27
Date Publish Online
2025-03-29