基于TurboID的植物蛋白邻近标记实验方法

doi:10.11983/CBB21104

摘要/Abstract

摘要：

邻近标记作为近些年发展起来的一项检测活细胞内蛋白互作关系和亚细胞结构蛋白组的新型技术, 已成功应用于多种动植物体系的研究。该技术通过给诱饵蛋白融合一个具有特定催化连接活性的酶, 在酶的催化作用下将小分子底物(如生物素)共价连接到酶邻近的内源蛋白, 通过富集和分析被标记的蛋白可获得与诱饵互作的蛋白组。经定向进化产生的生物素连接酶TurboID具有无蛋白毒性及催化效率高的优势。利用TurboID介导的邻近标记技术分析感兴趣蛋白的邻近蛋白组, 可研究细胞内瞬时发生或微弱的蛋白互作网络, 进而解析复杂的生物学过程。该文详细描述了在拟南芥(Arabidopsis thaliana)中基于TurboID的邻近标记实验方法及注意事项, 旨在为利用这一新技术研究植物蛋白互作关系提供参考。

关键词: TurboID, 邻近标记, 生物素, 蛋白互作

Abstract:

Proximity labeling (PL), a recently developed technique to detect protein-protein interactions and subcellular structural proteomes in living cells, has been successfully applied in various animal and plant systems. Proximity labeling is conducted by fusing an engineered enzyme with catalytic activity to a protein of interest (bait protein). With the catalysis of the enzyme, small molecular substrates such as biotin are covalently linked to endogenous proximal proteins, which can be further enriched and analyzed to identify the interactome of the bait protein. TurboID, a biotin ligase produced by directed evolution, has the advantages of non-toxicity and high catalytic efficiency. Using TurboID-based proximity labeling to analyze proximal proteins of bait proteins, we can study transient or weak protein interactions, which helps to understand the complex biological processes occurring inside cells. Here, we describe methods and related tips for TurboID-based proximal labeling in Arabidopsis thaliana, and hope to provide a reference for studying plant protein-protein interactions.

Key words: TurboID, proximity labeling, biotin, protein interactions

邝嘉怡, 李洪清, 沈文锦, 高彩吉. 基于TurboID的植物蛋白邻近标记实验方法. 植物学报, 2021, 56(5): 584-593.

Jiayi Kuang, Hongqing Li, Wenjin Shen, Caiji Gao. Methods for TurboID-based Proximal Labeling in Plants. Chinese Bulletin of Botany, 2021, 56(5): 584-593.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB21104

https://www.chinbullbotany.com/CN/Y2021/V56/I5/584

图/表 6

图1 基于TurboID的植物蛋白邻近标记实验流程

Figure 1 Experimental procedure of TurboID-based proximal labeling in plant

表1 实验所用相关试剂配方

Table 1 Related reagent formulations used in experiment

Solution	Composition	Working concentration
Protein Extraction Buffer	Tris-HCl (pH7.5)	50 mmol∙L^-1
	NaCl	150 mmol∙L^-1
	Sodium deoxycholate	0.5% (w/v)
	SDS	0.1% (w/v)
	EDTA	1 mmol∙L^-1
	Triton X-100	1% (v/v)
	DTT	1 mmol∙L^-1
	PMSF	1 mmol∙L^-1
	Leupeptin	10 μg∙mL^-1
	Protease Inhibitor Cocktail	1×
4×SDS Sample Buffer	Tris-HCl (pH6.8)	200 mmol∙L^-1
	SDS	8% (w/v)
	Glycerol	40% (v/v)
	β-mercaptoethanol	20% (v/v)
	Bromophenol blue	0.1% (w/v)
1×PBS buffer (pH7.4)	NaH₂PO₄∙2H₂O	0.263 g∙L^-1
	Na₂HPO₄∙12H₂O	1.856 g∙L^-1
	NaCl	8 g∙L^-1
	KCl	0.2 g∙L^-1
1×PBST	1×PBS buffer containing 0.05% Tween-20
Biotin stock (stored at -20°C)	Dissolve biotin in DMSO to a final concentration of 50 mmol∙L^-1

图2 稳定转化拟南芥中TurboID-EYFP-ATG8a的亚细胞定位将6日龄转TurboID-EYFP-ATG8a拟南芥幼苗转移至含1 µmol∙L-1 Conc A的MS缺氮液体培养基(MS-N)处理8小时, 在激光共聚焦荧光显微镜下观察亚细胞定位。Bars=50 μm

Figure 2 Subcellular localization of TurboID-EYFP-ATG8a in stable Arabidopsis lines 6-day-old transgenic Arabidopsis seedlings overexpressing TurboID-EYFP-ATG8a were transferred to nitrogen (N)- deficient medium with 1 µmol∙L-1 Conc A for 8 h followed by confocal microscopic analysis. Bars=50 μm

图3 生物素标记蛋白的免疫印迹检测 (A) 对野生型(WT)、转TurboID-EYFP (TY/WT)和转TurboID-EYFP-ATG8a (TY-8A/WT)拟南芥幼苗分别进行生物素处理(+, 50 µmol∙L-1, 3小时)或不进行处理(-), 使用SA-HRP与anti-GFP抗体进行免疫印迹检测, 分析TurboID-EYFP的活性与表达水平; (B) 对不同株系的转TurboID-EYFP (TY/WT)拟南芥幼苗进行生物素处理(+, 50 µmol∙L-1, 3小时)或不进行处理(-), 进行免疫印迹检测。SA-HRP: 辣根过氧化物酶标记链霉亲和素; CBB: 考马斯亮蓝

Figure 3 Immunoblot analysis of biotinylated protein (A) Wild-type (WT), transgenic Arabidopsis seedlings overexpressing TurboID-EYFP (TY/WT) or TurboID-EYFP-ATG8a (TY-8A/WT) were treated with 50 µmol∙L-1 biotin (+) for 3 h or 0 (-), activity and expression of TurboID-EYFP construct were analyzed by immunoblots with SA-HRP and anti-GFP antibodies; (B) Different lines of transgenic Arabidopsis seedlings overexpressing TurboID-EYFP (TY/WT) were treated with 50 µmol∙L-1 biotin (+) for 3 h or 0 (-), followed by immunoblots. SA-HRP: Streptavidin-HRP; CBB: Coomassie Brilliant Blue

图4 邻近标记中生物素浓度与处理时间的优化 (A) 使用不同浓度的生物素溶液分别对野生型和转TurboID-EYFP拟南芥处理1小时, 使用SA-HRP和anti-GFP抗体进行免疫印迹检测, 分析TurboID-EYFP的活性与表达水平; (B) 用50 µmol∙L-1生物素溶液处理野生型和转TurboID-EYFP拟南芥不同时间, 然后抽提蛋白并进行免疫印迹分析。SA-HRP: 辣根过氧化物酶标记链霉亲和素; CBB: 考马斯亮蓝

Figure 4 The optimization of biotin concentration and labeling time used in proximity labeling (A) Wild-type (WT) and transgenic Arabidopsis seedlings overexpressing TurboID-EYFP were treated with different concentrations of biotin solution for 1 h, followed by immunoblots with SA-HRP and anti-GFP antibodies to detect the proximity ligation activity and protein expression level of TurbolD-EYFP; (B) Wild-type (WT) and transgenic Arabidopsis seedings overexpressing TurboID-EYFP were treated with 50 µmol∙L-1 biotin for different times, followed by immunoblots with appropriate antibodies. SA-HRP: Streptavidin-HRP; CBB: Coomassie Brilliant Blue

图5 免疫印迹检测被磁珠富集的生物素化蛋白对表达TurboID-EYFP-ATG8a的拟南芥植株进行生物素处理(50 µmol∙L-1, 3小时), 提取蛋白后使用脱盐柱进行蛋白脱盐, 将脱盐后的样品等体积分成2份, 分别与20 μL Streptavidin Magnetic Beads过夜孵育, 最后分别用20 μL或200 μL 4× SDS Sample Buffer进行洗脱。Extract: 离心后的蛋白上清; After Desalting Column: 脱盐后的蛋白样品; Flow-through 1/2: 与链霉亲和素磁珠过夜孵育后的上清; Eluate 1: 使用20 μL buffer洗脱的蛋白样品; Eluate 2: 使用200 μL buffer洗脱的蛋白样品; SA-HRP: 辣根过氧化物酶标记链霉亲和素

Figure 5 Immunoblotting analyses of the biotinylated proteins enriched by Streptavidin Magnetic Beads Arabidopsis seedlings overexpressing TurboID-EYFP-ATG8a were treated with 50 µmol∙L-1 biotin for 3 hours. The protein was extracted and desalted through Desalting Column. The desalted sample was split into two parts and incubated with 20 μL Streptavidin Magnetic Beads overnight, followed by protein elution with 20 μL or 200 μL 4× SDS Sample Buffer, respectively. Extract: Protein supernatant after centrifugation; After Desalting Column: Protein sample after desalting; Flow-through 1 or 2: Supernatant after overnight incubation with streptavidin (SA) beads; Eluate 1: Protein sample eluated with 20 μL buffer; Eluate 2: Protein sample eluated with 200 μL buffer; SA-HRP: Streptavidin-HRP

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