Document Type: Research Paper
Department of Biological Sciences,BITS Pilani KK Birla Goa Campus,Zuari Nagar, Goa
Department of Biological Sciences, BITS PILANI KK BIRLA GOA CAMPUS ZUARI NAGAR GOA
Background: The chaperone activity of M. tb Acr is an important function that helps to prevent misfolding of protein substrates inside the host, especially in conditions of hypoxia.
Objectives: The aim of this study was to establish the correlation of structure and function of oligomerized recombinant Acr proteins after gel filtration chromatography.
Materials and Methods: Mycobacterium tuberculosis acr gene was cloned with an N-terminal His-tag in pET28a and expressed with IPTG induction in E. coli BL21DE3 cells. The activity of a recombinant construct of Acr in E. coli pET28a was checked by preventing thermal aggregation of citrate synthase at 45°C and chaperone activity against insulin B chain aggregation at 60ºC and 37°C. On further purification using gel filtration chromatography resin Sephacryl S-200, the protein was again tested for chaperone activity using insulin as substrate at 37°C with two types of samples without and with gel filtration designated A and B respectively. The effect of pre–heat treatment at 37°C and 60 °C on chaperone activity of both A and B samples were checked.
Results: The level of expression was 40 to 50 mg per litre. The protein was expressed in a soluble form at 37°C and purified by a 3 step gradient of imidazole using Ni-NTA resin. Gel filtration showed a mixture of 12 mers. Native-PAGE analysis showed a large proportion of monomers in the non-gel filtered sample Pre-heat treatment improved activity only after gel filtration.
Conclusions: The larger proportion of monomers in the non-gel filtered sample could be used to explain the difference in activity as compared to the gel-filtered samples in terms of molecular interaction with insulin. Increased oligomer size favorably affected secondary structure, a finding not reported so far, and warranting further investigation. A molecular level interaction of inhibition was predicted using Avogadro number of molecules and oligomer size.The difference in activity after pre –heat treatment seemed to indicate an important role for oligomerisation.