Cell and Tissue Journal

Abstract Aim: The role of Hsp70 chaperone from Rutilus frisii kutum in the thermal inactivation of luciferase in E. coli cell carrying Hsp70 and firefly luciferase was investigated.
Material and Methods: Co-transformation of E. coli cell was carried out with two expression vectors containing Hsp70 and firefly luciferase. The co-transformed cells carrying Hsp70 and luciferase were expressed under optimum conditions. After adding tetracycline, the cells were then incubated for 60 min at 40, 42, 44, 46, 48 and 50°C treatments. Finally, the luminescence activity of samples was calculated.
Results: After 30 min at 40 and 42°C temperatures, the luciferase activity in control samples reached almost zero, while in the co-transformed samples, about 72% and 60% of the luminance activity maintained compared with control samples (before heat treatment), and even after 60 min, up to 36% and 22% of the activity remained. Also, at 44 and 46°C temperatures in the initial times after stress, a significant difference was observed between the luciferase activity of the co-transformed and the control samples. In contrast, at 48 and 50°C temperatures, the changes of the luciferase activity of co-transformed samples were small compared with control samples even in the early stages of stress.
Conclusion: The thermal aggregation of luciferase as a significant reporter protein is inhibited at high temperatures via the activity of Hsp70 in the bacterial cell, which this process can be widely used in the food and pharmaceutical industries and the providing cancer diagnostic kits.
 

Abstract Aim: The aim of the present study was to investigate the optimal expression of Hsp70 recombinant Rutilus FrissiKutum liver in E. coli.
Material and methods: To evaluating of expression level of recombinant Hsp70, transformed E. coli were cultured in LB medium at 2, 4, 8, 12, 24, 36 and 48 hours, 20, 25, 30, 37 and 50 °C, extreme pH (5 and 9) and in present of heavy metals e.g. mercury, cobalt, iron, zinc and etc. Expression of recombinant Hsp70 protein was determined by SDS-PAGE 12% analysis of E. coli extracts followed by staining with Coomassie Blue.
Results: Optimum expression of recombinant Hsp70 was obtained at 4 and 8 h, 37 °C and pH 5. Additionally, the maximum and minimum expression of this protein was achieved in present of manganese and cobalt respectively. The results further indicate that the survival of E. coli with Hsp70 was enhanced compared to the control cells.
Conclusion: All results reveal that transformed bacteria are able to survive against extreme environmental conditions compared to control sample, because of having recombinant Hsp70. This heat-shock protein protects vital proteins from denaturing by its chaperone property. So, growth condition described in this study can be used for optimizing production of recombinant Hsp70 in E. coli host.