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. 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 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 cells are unable to support λ 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 cells grow adequately at temperatures ranging from 30 °C to 37 °C, but they die at elevated temperatures (>40 °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.
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 (BB2393 [C600 dnaK103(Am) thr::Tn10]) and E. coli dnaK756. E. coli dnaK103 cells produce a truncated DnaK that is non-functional, and as such, the cells grow adequately at 30 °C, while the strain is sensitive to cold and heat stress12,13. Similarly, the E. coli dnaK756/BB2362 (dnaK756 recA::TcR Pdm1,1) strain does not grow above 40 °C14,15. The E. coli dnaK756 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 λ DNA14. Additionally, E. coli 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 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.
1. Transformation
NOTE: Use sterile glassware for culture, pipette tips, and freshly prepared and autoclaved media. Prepare cultures of the E. coli cells in 2x yeast tryptone (YT) [1.6% tryptone (w/v), 1% yeast extract (w/v), 0.5% NaCl (w/v), 1.5% agar (w/v)] agar. General reagents used in the protocol and their sources are provided in the Table of Materials.
2. Cell plating
3. Confirming expression of recombinant proteins
4. SDS-PAGE and western blot analyses
Figure 2 presents an image of the scanned agar containing cells that were spotted and cultured at the permissive growth temperature of 37 °C and 43.5 °C, respectively. On the right-hand side of Figure 2, excised western blot components represent the expression of DnaK, KPf, and KPf-V436F in E. coli dnaK756 cells. As expected, all the E. coli dnaK756 cells cultured at the permissive growth temperature of 37 °C managed to grow. However, under the non-permissive growth conditions of 43.5 °C, only cells heterologously expressing DnaK and KPf managed to grow, as previously reported6,9,20(Figure 2). On the other hand, cells expressing KPf-V436F only grew at 37 °C but failed to grow at 43.5 °C. This demonstrates that DnaK and KPf were able to restore the growth defect of E. coli dnaK756 cells under heat stress conditions. The failure of cells heterologously expressing KPf-V436F to support the growth of cells at 43.5 °C demonstrates the lack of chaperone function of this protein. In this regard, KPf-V436F serves as an ideal negative control protein.
Figure 1: Principle of the complementation assay using E. coli dnaK756 cells to study the chaperone function of heterologously expressed proteins. E. coli dnaK756 expresses a native DnaK756 protein that is unable to protect the cells against heat stress. Introduction of functional heterologous Hsp70 rescues the cells from death upon exposure to heat stress. Please click here to view a larger version of this figure.
Figure 2: Complementation plate assay demonstrating the capabilities of DnaK and KPf to protect E. coli dnaK756 cells against heat stress. The transformed cells were cultured at 37 °C (permissive growth temperature) and 43.5 °C (non-permissive growth temperature). The cells were standardized and plated as serial dilutions. 'N' symbolizes 'Neat,' representing the first spot composed of undiluted cells. On the extreme right-hand side are the western blot excisions representing expression of the three proteins. Please click here to view a larger version of this figure.
The protocol demonstrates the utility of E. coli dnaK756cells in exploring the chaperone function of heterologously expressed Hsp70. This assay could be adopted to screen inhibitors targeting Hsp70 function in cellulo. However, one limitation of this method is that Hsp70s unable to substitute for DnaK in E. coli are not compatible with this assay. Lack of post-translational modification21 of some non-native Hsp70s may account for their lack of function within the E. coli system. A yeast-based complementation assay22 may address some of the shortcomings of the E. coli-based assay.
Several key steps are crucial to ensuring reproducible results. These include ensuring that only cells transformed by the respective plasmid construct are used during plating. In addition, it is important to avoid contamination of the culture throughout the steps. Furthermore, since stressed E. coli DnaK cells are susceptible to excessive filamentation10, it is important to avoid vigorous shaking during culture, as this physical strain promotes extensive filamentation. Excessively filamented cells give higher false apparent growth readings at OD600, leading to an overestimation of culture growth and adversely impacting cell standardization prior to plating. Although Hsp70 is not essential for E. coli growth under normal conditions, cells lacking this protein are stress susceptible10. For this reason, E. coli dnaK756 cells grow much slower after being transformed or during their recovery from glycerol stocks. Additionally, they are extremely sensitive to temperature fluctuations. Therefore, it is important to avoid opening the incubator door once the plated agar plates are placed inside until it is time to view the plates.
The western blot data are important to confirm the expression of the respective recombinant Hsp70 proteins in E. coli dnaK756 cells. Failure to express the respective protein leads to a false-negative result for cells growing under non-permissive growth temperature.
The authors have nothing to disclose.
The work was supported with grant funding obtained from the International Centre for Genetic Engineering and Biotechnology (ICGEB) grant number, HDI/CRP/012, Research Directorate of the University of Venda, grant I595, Department of Science and Innovation (DSI) and the National Research Foundation (NRF) of South Africa (grant numbers, 75464 & 92598) awarded to AS.
2-β-Mercaptoethanol | Sigma-Aldrich | 8,05,740 | Constituent for sample loading dye |
Acetic acid | Labchem | 101005125 | Constituent of destainer |
Acrylamide | Sigma-Aldrich | 8008300100 | Component of SDS |
Agar | Merck | HG000BX1.500 | Constituent of medium and liquid growth assay |
Agarose | Clever Scientific | 14131031 | Certified molecular biology agarose |
Ammonium persulfate | Sigma-Aldrich | 101875295 | Constituent for SDS-PAGE gel |
Ampicillin | VWR International | 0339—EU—25G | Selective antibiotic |
Bis | Sigma-aldrich | 1015460100 | Component of SDS |
Bromophenol | Sigma-Aldrich | 0449-25G | Constituent for sample loading dye |
CaCl2 | Sigma-Aldrich | 10043-52-4 | For competent cells preparation |
Coomassie brilliant blue | VWR International | 443293X | SDS-PAGE dye |
Dibasic sodium phosphate | Sigma-Aldrich | RB10368 | Constituent of PBS buffer |
ECL | Thermofischer Scientific | 32109 | Western blot detection reagent |
Ethidium Bromide | Thermofischer Scientific | 17898 | DNA intercalating dye |
Glycerol | Merck | SAAR2676520L | Constituent for sample loading dye |
Glycine | VWR International | 10119CU | Component of SDS |
IPTG | Glentham life sciences | 162IL | inducer |
Kanamycin | Melford | K0126 | Selective antibiotic |
Magnesium Chloride | Merck | SAAR4123000EM | Constituent of medium and liquid growth assay |
Methanol | Labchem | 113140129 | Constituent of destainer |
Monobasic potassium phosphate | Merck | 1,04,87,30,250 | Constituent of PBS buffer |
Peptone | Merck | HG000BX4.250 | Constituent of medium and liquid growth assay |
Potassium chloride | Merck | SAAR5042020EM | Constituent of PBS buffer |
PVDF membrane | Thermofischer scientific | PB7320 | Western blot membrane |
Sodium Chloride | Merck | SAAR5822320EM | Constituent of medium and liquid growth assay |
Sodium dodecyl sulphate | VWR International | 108073 | To resolve expressed proteins |
Spectramax iD3 | Separations | 373705019 | Automated plate reader |
TEMED | VWR international | ACRO420580500 | Component of SDS gel |
Tetracycline | Duchefa Biochemies | T0150.0025 | Selective antibiotic |
Tris | VWR International | 19A094101 | Component of SDS gel |
Tween20 | Merck | SAAR3164500XF | Constituent for Western wash buffer |
Western transfer chamber | Thermofisher Scientific | PB0112 | Transfer of protein to nitrocellulose membrane |
Yeast extract | Merck | HG000BX6.500 | Constituent of medium and liquid growth assay |
α-DnaK antibody | Inqaba | BK CAC09317 | Primary antibody |
α-rabbit antibody | Thermofischer scientific | 31460 | Secondary antibody |