Manipulating subcellular protein localization to enhance target protein accumulation in minicells.

IF 5.7 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Junhyeon Park, Karen M Polizzi, Jongmin Kim, Juhyun Kim
{"title":"Manipulating subcellular protein localization to enhance target protein accumulation in minicells.","authors":"Junhyeon Park, Karen M Polizzi, Jongmin Kim, Juhyun Kim","doi":"10.1186/s13036-025-00495-y","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"27"},"PeriodicalIF":5.7000,"publicationDate":"2025-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11955136/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Biological Engineering","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1186/s13036-025-00495-y","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

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.

操纵亚细胞蛋白定位以增强靶蛋白在小细胞中的积累。
背景:微细胞是细菌通过不规则细胞分裂形成的无染色体衍生物。与简化结构不同,微细胞保留了除染色体外的所有亲本细胞的细胞成分。这一特点减少了免疫原性反应,使其有利于各种生物技术应用,包括化学生产和药物输送。为了有效地利用微细胞,必须确保靶蛋白在其内部的积累,提高其作为递送载体的效率。结果:在本研究中,我们通过删除minCD基因来改造大肠杆菌,产生产生微细胞的菌株,并研究了增强微细胞内蛋白质积累的策略。比较蛋白质组学分析显示,微细胞保留了大多数亲本细胞的蛋白质,但表现出不对称的蛋白质组分布,导致选择性蛋白质富集。我们证明,无论表达水平如何,外源蛋白,如GFP和RFP,在小细胞中比在亲本细胞中积累得更丰富。为了进一步增强这种积累,我们通过将靶蛋白融合到极性定位信号来操纵蛋白质定位。与PtsI和Tsr融合的蛋白的积累量分别增加2.6倍和2.8倍,而与外源PopZ蛋白融合的蛋白在低诱导条件下的积累量增加了15倍。结论:这些发现强调了空间蛋白质组织在增强微细胞装载能力方面的关键作用。通过利用极性定位信号,这项工作为优化微型细胞作为各种应用的有效载体提供了一个强大的框架,从治疗递送到工业生物制造。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Journal of Biological Engineering
Journal of Biological Engineering BIOCHEMICAL RESEARCH METHODS-BIOTECHNOLOGY & APPLIED MICROBIOLOGY
CiteScore
7.10
自引率
1.80%
发文量
32
审稿时长
17 weeks
期刊介绍: Biological engineering is an emerging discipline that encompasses engineering theory and practice connected to and derived from the science of biology, just as mechanical engineering and electrical engineering are rooted in physics and chemical engineering in chemistry. Topical areas include, but are not limited to: Synthetic biology and cellular design Biomolecular, cellular and tissue engineering Bioproduction and metabolic engineering Biosensors Ecological and environmental engineering Biological engineering education and the biodesign process As the official journal of the Institute of Biological Engineering, Journal of Biological Engineering provides a home for the continuum from biological information science, molecules and cells, product formation, wastes and remediation, and educational advances in curriculum content and pedagogy at the undergraduate and graduate-levels. Manuscripts should explore commonalities with other fields of application by providing some discussion of the broader context of the work and how it connects to other areas within the field.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信