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

Escherichia coli dnaK756 세포를 사용한 Hsp70 Chaperone 활성 평가

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.

저자 스포트라이트: 말라리아 및 결핵 감염에서 열 충격 단백질 탐구

열충격 단백질 70의 샤페론 기능을 연구하기 위한 대장균 기반 보완 분석

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.

형질전환된 대장균 단백질의 SDS PAGE 및 웨스턴 블롯 분석을 사용한 Hsp70 샤페론 활성 분석

먼저 형질전환된 대장균 세포 80마이크로리터를 바이알로 피펫팅합니다. 피펫 20 마이크로리터의 4배 Laemmli SDS 로딩 버퍼를 바이알에 넣습니다. 가열 블록에서 서스펜션을 섭씨 100도에서 10분 동안 끓입니다.그런 다음 10마이크로리터의 샘플을 프리캐스트 SDS 겔에 로드합니다. SDS 러닝 버퍼에서 120볼트로 1시간 동안 실온에서 겔을 전기영동합니다. 전기영동이 완료되면 Coomassie 염색에 겔을 1시간 동안 염색합니다.증류수에 50% 메탄올과 10% 아세트산을 함유한 탈색 완충액에서 2시간 동안 겔을 탈색합니다. 겔을 겔 이미징 시스템에 넣어 단백질 밴드를 시각화합니다. 다음으로, SDS 겔의 단백질을 니트로셀룰로오스 막으로 옮긴다.세척 완충액에서 니트로셀룰로오스 멤브레인을 세 번 세척합니다. 1에서 2, 000의 비율로 묽게 된 항체를 포함하는 5%non-fat 우유에 막을 옮긴다. 항체가 있는 막을 섭씨 4도에서 60RPM으로 1시간 동안 흔들면서 배양합니다.다음으로, 니트로셀룰로오스 멤브레인을 TBS 트윈 완충액에서 각각 15분 동안 3회 세척하여 비특이적으로 결합된 항체를 제거합니다. 이제 멤브레인을 2차 항체가 있는 접시로 옮깁니다. 멤브레인을 섭씨 4도에서 60RPM으로 1시간 동안 흔들면서 배양합니다.이전과 같이 멤브레인을 세척한 후 향상된 화학발광 검출 시약을 사용하여 밴드를 분해합니다. 겔 이미저를 사용하여 밴드를 시각화합니다.

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

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JoVE Journal

Biochemistry

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

189 조회수.  University of Venda. 먼저 형질전환된 대장균 세포 80마이크로리터를 바이알로 피펫팅합니다. 피펫 20 마이크로리터의 4배 Laemmli SDS 로딩 버퍼를 바이알에 넣습니다. 가열 블록에서 서스펜션을 섭씨 100도에서 10분 동안 끓입니다.그런 다음 10마이크로리터의 샘플을 프리캐스트 SDS 겔에 로드합니다. SDS 러닝 버퍼에서 120볼트로 1시간 동안 실온에서 겔을 전기영동합니다. 전기영동이 완료되면 Co...

비디오: 형질전환된 대장균  단백질의 SDS PAGE 및 웨스턴 블롯 분석을 사용한 Hsp70 샤페론 활성 분석

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. ...