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FAST是一种基于分裂GFP的方法,用于检测在无细胞表达系统中合成的蛋白质溶液中的蛋白质

Thuy Duong Pham¹, Chiara Poletti¹, Therese Manuela Nloh Tientcheu¹

¹Laboratory of Genetics of Microorganisms and Microbial Biotechnology, School of Biosciences and Veterinary Medicine, University of Camerino, 62032, Camerino, MC, Italy.

Scientific reports

|April 5, 2024

概括

一种名为FAST的新方法简化了无细胞蛋白合成 (CFPS) 系统中的蛋白质检测. 这种光检测允许快速,安全和高效地监测合成的蛋白质及其净化.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
合成生物学合成生物学

背景情况:

无细胞蛋白合成 (CFPS) 是一种强大的工具,但由于成本,复杂性和危险性,传统的蛋白质检测方法往往是不切实际的.
现有的检测技术,如放射性标记,光标记和电泳,对于常规的CFPS应用有显著的局限性.
显然需要简化,可访问和安全的方法来检测通过CFPS产生的蛋白质.

研究的目的:

开发一种新的,简化的方法来检测在无细胞蛋白质合成 (CFPS) 系统中合成的蛋白质.
为了实现快速,高效,非危险的检测和在体外合成的蛋白质的潜在净化.
创建适用于各种基因和CFPS平台的多功能测试.

主要方法:

开发了FAST (用于翻译测试的分割-GFP的光组合),使用分割的GFP片段 (GFP1-10和GFP11).
融合了GFP11标签以准基因在CFPS系统中的表达.
在与MBP:GFP1-10相互作用时通过光检测到合成的蛋白质:GFP11的融合,使检测和可选的亲和力净化成为可能.

主要成果:

快速证明了光信号和合成蛋白质量之间的直接相关性,灵敏度为8±2 pmol.
光信号在合成4小时后达到平原.
该方法在自制和商业CFPS系统中成功检测了来自四个不同的大肠杆菌基因的蛋白质合成,并允许对抗生素对翻译的影响进行剂量依赖的研究.

结论:

FAST提供了一种快速,高效和安全的方法来检测CFPS系统中合成的蛋白质.
该试验具有多样性,可以与目标蛋白的亲和力净化相结合.
快速促进CFPS技术的更广泛的采用和应用.

关键词:

在CFPS中,CFPS是指CFPS.这是CspA.光是一种光效应.蛋白质合成蛋白质合成分拆-GFP的情况.

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