Purification, characterization and expression kinetics of heat shock protein 70 from Bubalus bubalis.
The present work on Bubalus bubalis (buffalo) was designed to study heat shock protein 70 (HSP70) induction in lymphocytes, its purification and characterization. HSP70 induction and expression kinetics at different temperatures and time durations were also studied. HSP70 purification was carried out by immunoaffinity chromatography using adenosine di-phosphate (ADP-agarose column) and the characterization of the purified protein was done using western blotting by mouse monoclonal anti-HSP70. The molecular weight of HSP70 of buffalo lymphocytes was found to be approximately of 68 kDa and was less than that of bovine brain HSP70. The purified HSP70 was assessed using indirect inhibition enzyme-linked immunosorbent assay (ELISA). A good amount of HSP70 (1430 eta g HSP70/100 microl) was recovered after purification, out of the total 2040.40 eta g of HSP70/100 microl of cell supernatant. To assess the impact of temperature and time dependent variability in the induction and expression pattern of HSP70, buffalo lymphocytes were subjected to three different temperature treatments, viz.: (I) 38 degrees C for 48 h and further exposed the same cells at 45 degrees C for 3 h, (II) 42 degrees C for 3 h, and (III) 45 degrees C for 3 h, respectively. The respective cell viability was found to be 68%, 63%, and 51%. The HSP70 levels were 58.30+/-4.37, 42.59+/-9.04 and 21.95+/-6.79 ng/million cells, respectively, at three temperature exposures. The results indicates that higher intensity and duration of temperature exposure cause higher HSP70 induction in buffalo lymphocytes to maintain cellular homeostasis with a threshold of thermal dose for maximum HSP70 expression. However immediate induction of HSP70 in the lymphocytes was dependent on magnitude of thermal exposure (stress level) and time of thermal exposure (stress duration). The present study on HSP70 and its induction will help likely to solve the problems related to the present scenario of thermo-adaptability in buffaloes.