Denaturation and refolding

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

Mammalian ferritins are heteropolymers of H- and L-chains. These subunits are very closely related, with an a-carbon rmsd of 0.5 A and 55% sequence identity conservation of primary sequence rises to 79% when considering those residues responsible for intersubunit interactions. Subunit assembly appears to take place via partially structured monomers associating to form fully structured homodimers, which then aggregate further. Upon chemical denaturation and refolding, heterodimers are rarely observed. ... 

The formation of inclusion bodies reflects a general and often intractable problem associated with expression of eukaryotic DNA s in prokaryotes. Even though significant amounts of insoluble protein can be isolated, solubilization by denaturation and refolding often results in low recoveries, particularly when refolding is attempted... 

Denaturation and Refolding of CK Denaturation and refolding methods were modifications of procedures us for refolding soluble CK isolated from tissue... 

Native % Activity" refers to CK samples which were incubated with extracts for three days, then assayed for loss of enzyme activity. "Refolded % Activity" refers to the same CK samples which were mixed with extracts and immediately denatured and refolded as described, then assayed for loss of activity at the same time as the native samples. CK activity values have been calculated as a percentage of the activity obtained for the native CK which was not incubated with octyl glucoside extract... 

The unfolding of a protein (i.e., disruption of the tertiary structure) is called denaturation. Reduction of disulfide bonds (Section 3.5) leads to even more extensive unraveling of the tertiary structure. Denaturation and reduction of disulfide bonds are frequently combined when complete disruption of the tertiary structure of proteins is desired. Under proper experimental conditions, the disrupted structure can then be completely recovered. This process of denaturation and refolding is a dramatic demonstration of the relationship between the primary structure of the protein and the forces that determine the tertiary structure. For many proteins, various other factors are needed for complete refolding, but the important point is that the primary structure determines the tertiary structure. 

We conclude this discussion with some recent data on denaturation and refolding of sperm whale myoglobin and apomyoglobin (Shen and Hermans (76)). Kinetic data on the refolding of this protein are potentially extremely valuable because a) the conformation of myoglobin is known, and b) the unfolding equilibrium is particularly well understood (34). The equilibrium follows a two state description, and, furthermore, free... 

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