Gopal Surajbhan Agrawal
National Institute of Pharmaceutical Education and Research, India
Posters & Accepted Abstracts: J Proteomics Bioinform
Recombinant therapeutic proteins when over-expressed in a suitable host like E. coli they tend to form aggregate of protein called as Inclusion Bodies (IB). IBs are inactive in nature but highly purified form of protein. IB formation can be in cytoplasmic or Periplasmic space. Mostly IB formation occurs in Cytosol. These IB are then isolated from host either by mechanical or chemical method. Isolated IB is purified using detergent. Purified IB is denatured using various denaturants and then refolded using various methods. Various traditional and Conventional method are attempted to refold the protein back to its active confirmation. Methods like dilution, dialysis, three phase partitioning (TPP), smart polymer affinity precipitation and nanoparticles are highly recommended method for refolding. In dilution and Dialysis, the protein is refolded by the gradual decreases in denaturing concentration and using refolding additives. In TPP, the protein is partitioned between an aqueous layer and organic layer using t-butanol as organic solvent and ammonium sulphate precipitation. Smart polymer precipitation uses the principle of stimuli sensitive polymer precipitation in response to stimulus. The polymer sensitive to a stimulus (pH; chemical) is incubated with protein (IB), the polymer is precipitated applying stimulus. Due to precipitation, the polymer forms complex with protein and the protein is refolded. Refolded protein is obtained by again applying the stimulus. In Nanoparticles, the principle is same as Smart Polymer, instead nanoparticles are used and there is no response to stimulus. The refolded protein is analysed for its specific activity against its specific substrate and compare with the natively expressed protein in order to know the extent of refolding.
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