A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity.
A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity.
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DOI：
10.1186/s12934-016-0474-y
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发表时间：
2016-05-03
影响因子：
6.4
通讯作者：
Nørholm MH
中科院分区：
文献类型：
--
作者：
Wendel S;Fischer EC;Martínez V;Seppälä S;Nørholm MH
细菌表面展示是生产细胞锚定功能蛋白和全细胞催化剂工程的一项有吸引力的技术。尽管各种外膜蛋白已用于表面展示，但仍缺少用于评估和开发表面展示系统的简单且通用的高通量兼容测定。使用对绿色荧光蛋白 (GFP) 具有高亲和力的单域抗体（也称为纳米抗体），我们构建了一个系统，可以对展示的蛋白质进行基于荧光的快速检测。外膜杂合蛋白LppOmpA和自转运蛋白C-IgAP以截然不同的效率将纳米抗体暴露在大肠杆菌表面。两个锚都能够功能性地展示酶几丁质酶 A 作为与纳米抗体的融合体，并且与单独展示纳米抗体相比，这显着增加了表达水平。我们使用流式细胞仪分析了单细胞与群体水平的显示能力，发现锚定信号肽对显示效率有很大影响。我们开发了一种廉价且易于读取的检测方法，用于使用纳米体：GFP 相互作用进行表面显示。该检测与最常见的荧光检测方法兼容，包括多孔板全细胞荧光检测、SDS-PAGE 凝胶荧光、显微镜检查和流式细胞术。我们预计该平台将有助于未来对蛋白质转运到活细胞表面的机制的深入研究，以及工业生物技术应用的优化。本文的在线版本 (doi:10.1186/s12934-016-0474-y) 包含补充材料，可供授权用户使用。
Bacterial surface display is an attractive technique for the production of cell-anchored, functional proteins and engineering of whole-cell catalysts. Although various outer membrane proteins have been used for surface display, an easy and versatile high-throughput-compatible assay for evaluating and developing surface display systems is missing. Using a single domain antibody (also called nanobody) with high affinity for green fluorescent protein (GFP), we constructed a system that allows for fast, fluorescence-based detection of displayed proteins. The outer membrane hybrid protein LppOmpA and the autotransporter C-IgAP exposed the nanobody on the surface of Escherichia coli with very different efficiency. Both anchors were capable of functionally displaying the enzyme Chitinase A as a fusion with the nanobody, and this considerably increased expression levels compared to displaying the nanobody alone. We used flow cytometry to analyse display capability on single-cell versus population level and found that the signal peptide of the anchor has great effect on display efficiency. We have developed an inexpensive and easy read-out assay for surface display using nanobody:GFP interactions. The assay is compatible with the most common fluorescence detection methods, including multi-well plate whole-cell fluorescence detection, SDS-PAGE in-gel fluorescence, microscopy and flow cytometry. We anticipate that the platform will facilitate future in-depth studies on the mechanism of protein transport to the surface of living cells, as well as the optimisation of applications in industrial biotech. The online version of this article (doi:10.1186/s12934-016-0474-y) contains supplementary material, which is available to authorized users.