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

Beoordeling van Hsp70-chaperonne-activiteit met behulp van Escherichia coli dnaK756-cellen

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.

Auteur in de schijnwerpers: onderzoek naar hitteschokeiwitten bij malaria- en tuberculose-infecties

Op Escherichia coli gebaseerde complementatietest om de chaperonnefunctie van heat shock-eiwit te bestuderen 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.

Analyse van Hsp70-chaperonne-activiteit met behulp van SDS PAGE- en Western Blot-analyses van getransformeerd E. coli-eiwit

Pipetteer om te beginnen 80 microliter getransformeerde E.coli-cellen in een injectieflacon. Pipetteer 20 microliter van viermaal Laemmli SDS-laadbuffer in de flacon. Kook de suspensie op 100 graden Celsius gedurende 10 minuten in een verwarmingsblok.Laad vervolgens 10 microliter van het monster op een geprefabriceerde SDS-gel. Elektroforeer de gel bij kamertemperatuur gedurende een uur bij 120 volt in SDS lopende buffer. Zodra de elektroforese is voltooid, kleurt u de gel gedurende een uur in de Coomassie-kleuring.Ontkleur de gel in een ontkleuringsbuffer met 50% methanol en 10% azijnzuur in gedestilleerd water gedurende twee uur. Plaats de gel in een gelbeeldvormingssysteem om de eiwitbanden te visualiseren. Breng vervolgens de eiwitten van de SDS-gel over naar een nitrocellulosemembraan.Was het nitrocellulosemembraan drie keer in de wasbuffer. Breng het membraan over in 5% magere melk die antilichamen bevat, verdund tot een verhouding van één op 2.000. Incubeer het membraan met de antilichamen bij vier graden Celsius terwijl je een uur lang schudt bij 60 RPM.Was vervolgens het nitrocellulosemembraan in TBS-tweenbuffer drie keer gedurende 15 minuten om niet-specifiek gebonden antilichamen te verwijderen. Breng nu het membraan over naar een schaal met het secundaire antilichaam. Incubeer het membraan bij vier graden Celsius terwijl je een uur lang op 60 RPM schudt.Gebruik na het wassen van het membraan zoals voorheen een verbeterd chemiluminescentiedetectiereagens om de banden op te lossen. Visualiseer de banden met behulp van een gelimager.

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

Transformatie van E. coli-cellen voor een complementatietest om de activiteit van Hsp70-chaperonne te beoordelen