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建立用于. 蛋白生产的蛋白分泌条件的双荧光素酶检测方法的开发。

Development of a split-luciferase assay to establish optimal protein secretion conditions for protein production by .

机构信息

Molecular Microbiology, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ, Amsterdam, The Netherlands.

Bacterial Cell Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands.

出版信息

Microbiology (Reading). 2024 Jun;170(6). doi: 10.1099/mic.0.001460.

DOI:10.1099/mic.0.001460
PMID:38847798
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11261832/
Abstract

is a Gram-positive bacterium that is frequently used in the bioindustry for the production of various proteins, because of its superior protein secretion capacities. To determine optimal conditions for protein secretion by , a quick and sensitive method for measuring protein secretion is crucial. A fast and universal assay is most useful for detecting diverse proteins in a high-throughput manner. In this study, we introduce a split-luciferase-based method for measuring protein secretion by . The NanoBiT system was used to monitor secretion of four different proteins: xylanase A, amylase M, protein glutaminase A, and GFP nanobody. Our findings underscore the split-luciferase system as a quick, sensitive, and user-friendly method.

摘要

是一种革兰氏阳性菌，由于其具有优越的蛋白质分泌能力，因此经常被用于生物工业生产各种蛋白质。为了确定蛋白质分泌的最佳条件，一种快速灵敏的蛋白质分泌测量方法至关重要。一种快速且通用的测定法最适合高通量检测多种蛋白质。在本研究中，我们引入了一种基于荧光素酶的方法来测量 的蛋白质分泌。NanoBiT 系统被用于监测四种不同蛋白质的分泌：木聚糖酶 A、淀粉酶 M、蛋白谷氨酰胺酶 A 和 GFP 纳米抗体。我们的研究结果强调了荧光素酶系统作为一种快速、灵敏和用户友好的方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/6b2cad12b0a5/mic-170-01460-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/ebbe367b9882/mic-170-01460-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/71c385e372b4/mic-170-01460-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/1bcc430d4e35/mic-170-01460-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/3bf590f11280/mic-170-01460-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/6b2cad12b0a5/mic-170-01460-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/ebbe367b9882/mic-170-01460-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/71c385e372b4/mic-170-01460-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/1bcc430d4e35/mic-170-01460-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/3bf590f11280/mic-170-01460-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/47a2/11261832/6b2cad12b0a5/mic-170-01460-g005.jpg

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Biosensor-Based Optimization of Cutinase Secretion by .基于生物传感器的[具体对象]角质酶分泌优化
Front Microbiol. 2021 Oct 28;12:750150. doi: 10.3389/fmicb.2021.750150. eCollection 2021.
3
Scaling production of GFP1-10 detector protein in E. coli for secretion screening by split GFP assay.
通过 GFP 片段分析检测筛选，对 GFP1-10 探测器蛋白在大肠杆菌中的分泌生产进行规模化。
Microb Cell Fact. 2021 Sep 30;20(1):191. doi: 10.1186/s12934-021-01672-6.
4
The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis.核糖体结合位点间隔序列的长度控制枯草芽孢杆菌细胞内和分泌蛋白的生产产量。
Microb Cell Fact. 2020 Jul 29;19(1):154. doi: 10.1186/s12934-020-01404-2.
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A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems.一种基于 NanoLuc 荧光素酶的检测方法，可实时分析细菌 III 型分泌系统的蛋白质分泌和注射。
Mol Microbiol. 2020 Jun;113(6):1240-1254. doi: 10.1111/mmi.14490. Epub 2020 Mar 4.
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