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

Valutazione dell'attività degli chaperoni Hsp70 utilizzando le cellule di 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.

Autore in primo piano: Esplorazione delle proteine da shock termico nelle infezioni da malaria e tubercolosi

Test di complementazione basato su Escherichia coli per studiare la funzione chaperone della proteina da shock termico 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.

Analisi dell'attività dello chaperone Hsp70 mediante analisi SDS PAGE e Western blot della proteina trasformata di E. coli

Per iniziare, pipettare 80 microlitri di cellule di E.coli trasformate in una fiala. Pipettare 20 microlitri di tampone di caricamento Laemmli SDS quattro volte nel flaconcino. Far bollire la sospensione a 100 gradi Celsius per 10 minuti in un blocco riscaldante.Quindi caricare 10 microlitri del campione su un gel SDS prefabbricato. Elettroforesi del gel a temperatura ambiente per un'ora a 120 volt in tampone di funzionamento SDS. Una volta completata l'elettroforesi, colorare il gel con la colorazione di Coomassie per un'ora.Decolorare il gel in un tampone decolorante contenente il 50% di metanolo e il 10% di acido acetico in acqua distillata per due ore. Posizionare il gel in un sistema di imaging del gel per visualizzare le bande proteiche. Successivamente, trasferire le proteine del gel SDS su una membrana di nitrocellulosa.Lavare tre volte la membrana di nitrocellulosa nel tampone di lavaggio. Trasferire la membrana al 5% di latte scremato contenente anticorpi diluiti in un rapporto di uno a 2.000. Incubare la membrana con gli anticorpi a quattro gradi Celsius agitando a 60 RPM per un'ora.Successivamente, lavare la membrana di nitrocellulosa nel tampone TBS per interpolazione tre volte per 15 minuti ciascuna per rimuovere eventuali anticorpi non legati in modo specifico. Ora trasferisci la membrana in un piatto con l'anticorpo secondario. Incubare la membrana a quattro gradi Celsius agitando a 60 RPM per un'ora.Dopo aver lavato la membrana come prima, utilizzare un reagente di rilevamento della chemiluminescenza potenziato per risolvere le bande. Visualizza le bande utilizzando un gel imager.

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 Views.  University of Venda. Per iniziare, pipettare 80 microlitri di cellule di E.coli trasformate in una fiala. Pipettare 20 microlitri di tampone di caricamento Laemmli SDS quattro volte nel flaconcino. Far bollire la sospensione a 100 gradi Celsius per 10 minuti in un blocco riscaldante.Quindi caricare 10 microlitri del campione su un gel SDS prefabbricato. Elettroforesi del gel a temperatura ambiente per un'ora a 120 volt in tampone di funzionamento SDS. Una volta completata l'elettroforesi, colorare il gel c...

Video: Analisi dell'attività dello chaperone Hsp70 mediante analisi SDS PAGE e Western blot della proteina trasformata di E. coli 

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. ...