增强重组蛋白质表面展示的新型细菌底盘

IF 2.3 4区 医学 Q3 BIOPHYSICS Cellular and molecular bioengineering Pub Date : 2024-09-13 DOI:10.1007/s12195-024-00819-w
Rui Zhang, Ningyuan Ye, Zongqi Wang, Shaobo Yang, Jiahe Li
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引用次数: 0

摘要

引言 细菌表面展示是一种将蛋白质和分子展示在细菌细胞外表面的重要生物技术。然而,它也有局限性,包括对宿主细菌的潜在毒性和显示效率的可变性。为了解决这些问题,我们研究了去除大肠杆菌中丰富的非必要外膜蛋白(OMPs)作为改善重组蛋白表面展示的新策略。我们在两种大肠杆菌菌株(K-12 MG1655 和大肠杆菌 BL-21)中成功敲除了 OmpA,这两种菌株具有广泛的研究和治疗应用价值。然后,我们将敲除 ompA 的菌株和两种 OMP 与三种治疗蛋白结合起来,包括抗毒素酶(ClbS)、用于激活细胞毒性 T 细胞的白细胞介素 18(IL-18)和用于阻断免疫检查点的抗 CTLA4 纳米抗体(αCTLA4)。结果 通过流式细胞仪测试了六种不同的显示构建物的显示水平,结果显示,与同源野生型菌株相比,oppA 基因敲除菌株提高了细菌中显示的百分比和水平。此外,治疗效果的提高也为基于活细菌的疗法提供了可能性,扩大了该技术在该领域的相关性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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A New Bacterial Chassis for Enhanced Surface Display of Recombinant Proteins

Introduction

Bacterial surface display is a valuable biotechnology technique for presenting proteins and molecules on the outer surface of bacterial cells. However, it has limitations, including potential toxicity to host bacteria and variability in display efficiency. To address these issues, we investigated the removal of abundant non-essential outer membrane proteins (OMPs) in E. coli as a new strategy to improve the surface display of recombinant proteins.

Methods

We targeted OmpA, a highly prevalent OMP in E. coli, using the lambda red method. We successfully knocked out ompA in two E. coli strains, K-12 MG1655 and E. coli BL-21, which have broad research and therapeutic applications. We then combined ompA knockout strains and two OMPs with three therapeutic proteins including an anti-toxin enzyme (ClbS), interleukin 18 (IL-18) for activating cytotoxic T cells and an anti- CTLA4 nanobody (αCTLA4) for immune checkpoint blockade.

Results

A total of six different display constructs were tested for their display levels by flow cytometry, showing that the ompA knockout strains increased the percentage as well as the levels of display in bacteria compared to those of isogenic wild-type strains.

Conclusions

By removing non-essential, highly abundant surface proteins, we develop an efficient platform for displaying enzymes and antibodies, with potential industrial and therapeutic applications. Additionally, the enhanced therapeutic efficacy opens possibilities for live bacteria-based therapeutics, expanding the technology’s relevance in the field.

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来源期刊
CiteScore
5.60
自引率
3.60%
发文量
30
审稿时长
>12 weeks
期刊介绍: The field of cellular and molecular bioengineering seeks to understand, so that we may ultimately control, the mechanical, chemical, and electrical processes of the cell. A key challenge in improving human health is to understand how cellular behavior arises from molecular-level interactions. CMBE, an official journal of the Biomedical Engineering Society, publishes original research and review papers in the following seven general areas: Molecular: DNA-protein/RNA-protein interactions, protein folding and function, protein-protein and receptor-ligand interactions, lipids, polysaccharides, molecular motors, and the biophysics of macromolecules that function as therapeutics or engineered matrices, for example. Cellular: Studies of how cells sense physicochemical events surrounding and within cells, and how cells transduce these events into biological responses. Specific cell processes of interest include cell growth, differentiation, migration, signal transduction, protein secretion and transport, gene expression and regulation, and cell-matrix interactions. Mechanobiology: The mechanical properties of cells and biomolecules, cellular/molecular force generation and adhesion, the response of cells to their mechanical microenvironment, and mechanotransduction in response to various physical forces such as fluid shear stress. Nanomedicine: The engineering of nanoparticles for advanced drug delivery and molecular imaging applications, with particular focus on the interaction of such particles with living cells. Also, the application of nanostructured materials to control the behavior of cells and biomolecules.
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