Refolding strategies

Hackenberger CP, Chen MM, Imperiali B. Expression of N-terminal Cys-protein fragments using an intein refolding strategy. Bioorg. Med. Chem. 2006 14(14) 5043-5048. 

Protein fragment complementation assays are based on an enzyme reassembly strategy whereby a protein-protein interaction promotes the efficient refolding and complementation of enzyme fragments to restore an active enzyme. The approach was initially developed using the reconstitution of ubiquitin as a sensor for protein-protein interactions (Johnsson and Varshavsky, 1994). Ubiquitin is a 76 amino acid protein that... 

The inherent drawbacks of the oxidative refolding approach for synthetic polypeptides containing multiple cysteine residues is the individual behavior of each peptide that derives from the encoded sequence, more or less pronounced structural information which prevents general procedures to be elaborated and proposed. Nevertheless, this synthetic approach remains attractive because of its simplicity compared to the synthetic strategies for re-gioselective disulfide bond formation (Section 6.1.1-6.1.4), and it is certainly indispensable if the number of cysteine residues exceeds the presently available chemistry for site-directed cysteine pairings. 

Unlike macroscopic machines, biomolecules constantly experience substantial fluctuations in their structures. Compared to the thermal energy, an individual nonco-valent bond is only marginally stable. Thus, biomolecules can partially unfold and refold if this dynamics is more effectively exploited to achieve their functional goal. Kinesins also adopt this strategy to effectively march along the MT. 

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