Inclusion bodies and purification of proteins in biologically active forms.
Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved.
Mukhopadhyay
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National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
D000818:Animals; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004220:Disulfides; D007328:Insulin; D011994:Recombinant Proteins; D011392:Proline; D008715:Methionine; D010959:Tissue Plasminogen Activator