analysis of hsp70 chaperone activity using sds page and western blot analyses of transformed e. coli protein

使用 大肠杆菌 dnaK756 细胞评估 Hsp70 伴侣活性

Heat shock proteins play an important role as molecular chaperones by facilitating protein folding, preventing protein aggregation, and reversing protein misfolding1,2. Heat shock protein 70 (Hsp70) is one of the most prominent molecular chaperones, playing a central role in protein homeostasis3,4. DnaK is the E. coli Hsp70 homologue5.
Various biophysical, biochemical, and cell-based assays have been developed to explore the chaperone activity of Hsp70 and to screen for inhibitors targeting this chaperone6,7,8. Hsp70 is a highly conserved protein. For this reason, several Hsp70s of eukaryotic organisms, such as Plasmodium falciparum (the main agent of malaria), have been reported to substitute for DnaK function in E. coli6,9. In this way, an E. coli-based complementation assay has been developed involving the heterologous expression of Hsp70s in E. coli to explore their cytoprotective function. Typically, this assay involves the utilization of E. coli cells that are either deficient for DnaK or that express a native DnaK that is functionally compromised. While DnaK is not essential for E. coli growth under normal conditions, it becomes essential when the cells are grown under stressful conditions such as elevated temperatures or other forms of stress10,11.
E. coli strains that have been developed to study Hsp70 function using a complementation assay include E. coli dnaK103&#160;(BB2393 [C600 dnaK103(Am) thr::Tn10]) and E. coli dnaK756. E. coli dnaK103&#160;cells produce a truncated DnaK that is non-functional, and as such, the cells grow adequately at 30 &#176;C, while the strain is sensitive to cold and heat stress12,13. Similarly, the E. coli dnaK756/BB2362 (dnaK756&#160;recA::TcR Pdm1,1) strain does not grow above 40 &#176;C14,15. The E. coli dnaK756&#160;strain expresses a mutant native DnaK (DnaK756) characterized by three glycine-to-aspartate substitutions at positions 32, 455, and 468, giving rise to compromised proteostatic outcomes. Consequently, this strain is resistant to bacteriophage &#955; DNA14. Additionally, E. coli&#160;dnaK756 exhibits elevated ATPase activity, while its affinity for the nucleotide exchange factor, GrpE, is reduced16. E. coli DnaK mutant strains serve as ideal models for investigating the chaperone activity of Hsp70 through a complementation approach. Since DnaK is only essential under stressful conditions, the complementation assay is typically conducted at elevated temperatures (Figure 1). Some advantages of using E. coli for this study include its well-characterized genome, rapid growth, and the low cost of culturing and maintenance17.
In this article, we describe in detail a protocol involving the use of E. coli dnaK756&#160;cells to study the function of Hsp70. The Hsp70s we employed in the assay are wild-type DnaK and its chimeric derivative, KPf (made up of the ATPase domain of DnaK fused to the C-terminal substrate-binding domain of Plasmodium falciparum Hsp70-16,18). KPf-V436F was heterologously expressed as a negative control since the mutation essentially blocks it from binding substrates, thus abrogating its chaperone activity9.

作者聚焦：探索疟疾和结核病感染中的热休克蛋白

基于大肠杆菌的互补测定研究热休克蛋白的伴侣功能 70

I study the role of heat shock proteins in conferring future static protection to antigens that cause human infections such as malaria and tuberculosis. My research essentially focuses on the structure and function features of the heat shock protein machinery of these pathogens that cause human infections. Heatstroke proteins are generally highly concept, however, they display some level of functional specialization.In addition, they are implicated in drug resistant. As such, there are ongoing efforts to target them towards reversing drug resistance in various disease models. They include biochemical, biophysical, bioinformatics, and cell biologic techniques.In other words, various areas of techniques are used to study the roles of these proteins. In our case, the study of proteins of Plasmodium was the main agent of malaria and that they are difficult to produce and this limits the capability to study these proteins. For example, it is difficult to generate sufficient levels to crystallize and image the proteins.We have established some of the functional networks of these proteins in the malaria parasite. We have also established assays to study the compounds targeting these proteins in agents causing malaria and TB.The current protocol is used to explore the chaperone protein folding role and then cytoprotective function of HSP 70 using equal as a model. The protocol could also be adopted for screening small molecule inhibitors targeting protein 70.

使用 SDS PAGE 分析 Hsp70 伴侣活性，并对转化 的大肠杆菌 蛋白进行蛋白质印迹分析

首先，将 80 微升转化的大肠杆菌细胞移液到小瓶中。将 20 微升 4 倍 Laemmli SDS 上样缓冲液移入小瓶中。将悬浮液在加热块中以 100 摄氏度煮沸 10 分钟。然后将 10 μL 样品加载到预制 SDS 凝胶上。在室温下将凝胶在SDS电泳缓冲液中以120伏电泳一小时。电泳完成后，在考马斯染色剂中染色凝胶一小时。在含有50%甲醇和10%乙酸的脱色缓冲液中，在蒸馏水中对凝胶进行脱色两小时。将凝胶放入凝胶成像系统中以可视化蛋白质条带。接下来，将SDS凝胶的蛋白质转移到硝酸纤维素膜上。在洗涤缓冲液中洗涤硝酸纤维素膜三次。将膜转移到含有抗体的 5% 脱脂牛奶中，该抗体稀释至 1 比 2， 000 的比例。将膜与抗体在4摄氏度下孵育，同时以60RPM振荡一小时。接下来，在 TBS 吐温缓冲液中洗涤硝酸纤维素膜 3 次，每次 15 分钟，以除去任何非特异性结合的抗体。现在将膜转移到装有二抗的培养皿中。将膜在4摄氏度下孵育，同时以60RPM振荡一小时。像以前一样洗涤膜后，使用增强型化学发光检测试剂来解析条带。使用凝胶成像仪可视化条带。

analysis-hsp70-chaperone-activity-using-sds-page-western-blot

Research

JoVE Journal

Biochemistry

Analysis of Hsp70 Chaperone Activity Using SDS PAGE and Western Blot Analyses of Transformed E. coli Protein

189 次观看.  University of Venda. 首先，将 80 微升转化的大肠杆菌细胞移液到小瓶中。将 20 微升 4 倍 Laemmli SDS 上样缓冲液移入小瓶中。将悬浮液在加热块中以 100 摄氏度煮沸 10 分钟。然后将 10 μL 样品加载到预制 SDS 凝胶上。在室温下将凝胶在SDS电泳缓冲液中以120伏电泳一小时。电泳完成后，在考马斯染色剂中染色凝胶一小时。在含有50%甲醇和10%乙酸的脱色缓冲液中，在蒸馏水中对凝胶进行脱色?...

视频: 使用 SDS PAGE 分析 Hsp70 伴侣活性，并对转化 的大肠杆菌  蛋白进行蛋白质印迹分析

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70&#160;genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756&#160;cells are unable to support &#955; bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756&#160;cells grow adequately at temperatures ranging from 30 &#176;C to 37 &#176;C, but they die at elevated temperatures (&#62;40 &#176;C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. ...

使用 SDS PAGE 分析 Hsp70 伴侣活性，并对转化 的大肠杆菌 蛋白进行蛋白质印迹分析

使用 SDS PAGE 分析 Hsp70 伴侣活性，并对转化的大肠杆菌蛋白进行蛋白质印迹分析