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

Оценка активности шаперона Hsp70 с использованием клеток Escherichia coli dnaK756

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.

В центре внимания автора: Изучение белков теплового шока при малярии и туберкулезе

Анализ комплементации на основе Escherichia coli для изучения шаперонной функции белка теплового шока 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.

Анализ активности шаперона Hsp70 с помощью SDS PAGE и вестерн-блоттинга трансформированного белка E. coli

Для начала пипеткой 80 мкл трансформированных клеток кишечной палочки во флакон. Пипетка 20 мкл четырехкратного загрузочного буфера Laemmli SDS во флакон. Прокипятите суспензию при температуре 100 градусов Цельсия в течение 10 минут в нагревательном блоке.Затем загрузите 10 микролитров образца на сборный гель SDS. Электрофорезируйте гель при комнатной температуре в течение одного часа при напряжении 120 вольт в работающем буфере SDS. После завершения электрофореза окрасьте гель красителем Coomassie в течение одного часа.Обесцвечивайте гель в обесцвечивающем буфере, содержащем 50% метанола и 10% уксусной кислоты, в дистиллированной воде в течение двух часов. Поместите гель в систему визуализации геля для визуализации белковых полос. Далее переносят белки геля SDS на нитроцеллюлозную мембрану.Промойте нитроцеллюлозную мембрану три раза в буфере для промывки. Перенесите мембрану на 5%-ное обезжиренное молоко, содержащее антитела, разбавленные в соотношении 1 к 2 000. Инкубируйте мембрану с антителами при температуре четыре градуса Цельсия при встряхивании при 60 оборотах в минуту в течение одного часа.Затем промойте нитроцеллюлозную мембрану в буфере TBS три раза по 15 минут каждый, чтобы удалить любые неспецифически связанные антитела. Теперь перенесите мембрану в чашку со вторичным антителом. Инкубируйте мембрану при температуре четыре градуса Цельсия при встряхивании при 60 оборотах в минуту в течение одного часа.После промывки мембраны, как и раньше, используйте улучшенный реагент для обнаружения хемилюминесценции, чтобы рассосать полосы. Визуализируйте полосы с помощью гелевого имидж-сканера.

Research

JoVE Journal

Biochemistry

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

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. ...

Transformation of E. coli Cells for a Complementation Assay to Assess Hsp70 Chaperone Activity

Трансформация клеток E. coli для анализа комплемента для оценки активности шаперона Hsp70