使用荧光光谱法测定蛋白质聚集抑制实验中分子伴侣的活性

Manish Bhuwan; Madhuri Suragani; Nasreen Z. Ehtesham; Seyed E. Hasnain

doi:10.21769/BioProtoc.2107

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry

使用荧光光谱法测定蛋白质聚集抑制实验中分子伴侣的活性

MB Manish Bhuwan ^*

MS Madhuri Suragani ^*§

Nasreen Z. Ehtesham email

Seyed E. Hasnain email

(*contributed equally to this work, §Deceased) 发布: 2017年01月20日第7卷第2期 DOI: 10.21769/BioProtoc.2107 浏览次数: 11869

评审: Valentine V TrotterSoazig Le Guyon Anonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of mBio, featuring study using the protocol.

Mar/Apr 2016

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

评估粘蛋白与细菌和细菌蛋白结合的实验

Lubov S. Grigoryeva [...] Nicholas P. Cianciotto

2021年03月05日 3874 阅读

从大肠杆菌中大规模纯化III型毒素-抗毒素核糖蛋白复合物及其成分，用于生物物理学研究

Parthasarathy Manikandan [...] Mahavir Singh

2023年07月05日 1031 阅读

使用 Spy Chemistry 进行抗体纯化

Xiaofeng Yang [...] Zisha Lao

2025年04月20日 1023 阅读

Abstract

The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway (Figure 1). Usually molecular chaperones exhibit differential affinities with different conformations of the substrate. With the exception of the sHsp family of molecular chaperones, the shift from a high-affinity binding state to the low-affinity release state is triggered by ATP binding and hydrolysis (Haselback and Buchner, 2015). Aggregation prevention assay is a simple, yet definitive assay to determine the chaperone activity of heat labile proteins such as Maltodextrin glucosidase (MalZ), Citrate Synthase (CS) and NdeI. This is based on the premise that proteins with chaperone like activity should prevent protein substrates (MalZ, CS and NdeI) from thermal aggregation. Here, we describe a detailed protocol for aggregation prevention assay using two different chaperone proteins, resistin and MoxR1, identified from our lab. Resistin, a human protein (hRes) and MoxR1 a Mycobacterium tuberculosis protein were analysed for their effect on prevention of MalZ/Citrate Synthase (CS)/NdeI aggregation.

Figure 1. Mechanism of action of molecular chaperones. Citrate synthase folds via increasingly structured intermediates (I₁, I₂) from the unfolded state (U) to the folded state (N). Under heat shock conditions, this process is reversed.

Keywords: Molecular chaperone (分子伴侣)

Thermal aggregation prevention (热凝聚预防)

ATPase activity assay (ATP酶活性测定)

GroEL (GroEL)

MoxR1 (MoxR1)

Resistin proteins (抵抗素蛋白)

Refolding of Enzymes & Proteins (酶和蛋白质的再折叠)

Background

To elucidate the chaperone activity of a protein in vitro, several methods have been developed. Primarily these methods are based on examining the enzyme function and the ability of the chaperone to refold and protect enzyme activity under heat or other stress conditions. Other method to identify and study chaperones includes in silico analysis, or co-purification with other proteins. The limitations with such methods are less reproducibility or inherent high chances of false positive results. In the current method the use of light scattering to detect prevention of protein aggregation relies on thermal stabilization of protein only from denatured state, and in the presence of a chaperone. In contrast, if aggregate is formed from native or intermediate state of the protein the amount of aggregation might increase thereby decreasing the chance of false positive. Furthermore, this method uses the purified recombinant protein for the assay and therefore, can also be used to study other chaperone proteins from other bacterial sources.

Materials and Reagents

Pipette tips
Microcentrifuge tube
Parafilm
E. coli BL21 (DE3) cells
Citrate synthase ammonium sulfate suspension from porcine heart (Sigma-Aldrich, catalog number: C3260 )
Ammonium sulphate
Tris (pH 8.0) (AMRESCO, catalog number: 0497 )
Luria Bertani agar plates
Ampicillin (Sigma- Aldrich, catalog number: A9518 )
Isopropyl β-D-1-thiogalactopyranoside, IPTG (Sigma-Aldrich, catalog number: I6758 )
DNAse I (Sigma-Aldrich, catalog number: AMPD1 )
Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M2670 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 7626 )
Bradford reagent (Bio-Rad Laboratories, catalog number: 5000006 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
Recombinant protein MoxR1 and hRes (1 mg/ml) (Purified in the laboratory)
Purified recombinant protein GroEL (0.5 mg/ml) (Purified in the laboratory)
Lysozyme (1 mg/ml) (Sigma-Aldrich, catalog number: 4919 )
Milli-Q water
NdeI (20,000 U/ml) (New England BioLab, catalog number: R0111 )
Maltodextrin glucosidase protein (1 mg/ml) (Purified in the laboratory)
EDTA (Sigma-Aldrich, catalog number: E9884 )
Sodium phosphate (dibasic) heptahydrate (Na₂HPO₄·7H₂O) (AMRESCO, catalog number: 0348 )
Sodium phosphate (monobasic) anhydrous (NaH₂PO₄) (AMRESCO, catalog number: 0571 )
Sodium chloride (NaCl) (AMRESCO, catalog number: 0241 )
Imidazole (AMRESCO, catalog number: 0527 )
Glycerol (AMRESCO, catalog number: 0854 )
TE buffer (see Recipes)
20 mM sodium phosphate buffer pH 7.4 (see Recipes)
Binding buffer (see Recipes)
Washing buffer (see Recipes)
Elution buffer (see Recipes)
Dialysis buffer (see Recipes)

Equipment

Pipette (Gilson, model: Pipetman Neo^®)
Centrifuge (Hermle Labor Technik, model: Z 326 K )
PerkinElmer spectrofluorometer (PerkinElmer, model: LS55 ) with controlled temperature peltier block
Incubator (Eppendorf, Brunswick^TM, model: 44/44R )
Quartz cuvette (PerkinElmer, Suprasil^®, catalog number: B0631071 )
pH meter (Thermo Fisher Scientific, Thermo Scientific, model: CyberScan Ph 510 )
Fluorescence spectrophotometer (Agilent Technologies, model: Cary Eclipse )

Software

Microsoft Excel
Graphpad Prism 5

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Bhuwan, M., Suragani, M., Ehtesham, N. Z. and Hasnain, S. E. (2017). Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry. Bio-protocol 7(2): e2107. DOI: 10.21769/BioProtoc.2107.