Chapter: Pharmaceutical Biotechnology: Fundamentals and Applications - Biophysical and Biochemical Analysis of Recombinant Proteins

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Protein Folding

Proteins become functional only when they assume a distinct tertiary structure.

PROTEIN FOLDING

Proteins become functional only when they assume a distinct tertiary structure. Many physiologically and therapeutically important proteins present their surface for recognition by interacting with molecules such as substrates, receptors, signaling proteins and cell–surface adhesion macromolecules. When recombinant proteins are produced in Escherichia coli, they often form inclusion bodies into which they are deposited as insoluble proteins. Formation of such insoluble states does not naturally occur in cells where they are normally synthesized and transported. Therefore, an in vitro process is required to refold insoluble recombinant proteins into their native, physiologically active state. This is usually accom-plished by solubilizing the insoluble proteins with detergents or denaturants, followed by the purifica-tion and removal of these reagents concurrent with refolding the proteins.

Unfolded states of proteins are usually highly stable and soluble in the presence of denaturing agents. Once the proteins are folded correctly, they are also relatively stable. During the transition from the unfolded form to the native state, the protein must go through a multitude of other transition states in which it is not fully folded and denaturants or solubilizing agents are at low concentrations or even absent.

The refolding of proteins can be achieved in various ways. The dilution of proteins at high denaturant concentration into aqueous buffer will decrease both denaturant and protein concentration simultaneously. The addition of an aqueous buffer to a protein–denaturant solution also causes a decrease in concentrations of both denaturant and protein. The difference in these procedures is that, in the first case, both denaturant and protein concentrations are the lowest at the beginning of dilution and gradually increase as the process continues. In the second case, both denaturant and protein concentrations are high-est at the beginning of dilution and gradually decrease as the dilution proceeds. Dialysis or the diafiltration of protein in the denaturant against an aqueous buffer resembles the second case, since the denaturant concentration decreases as the procedure continues. In this case, however, the protein concen-tration remains unchanged. Refolding can also be achieved by first binding the protein in denaturants to a solid phase, i.e., to a column matrix, and then equilibrating it with an aqueous buffer. In this case,protein concentrations are not well defined. Each procedure has advantages and disadvantages and may be applicable for one protein, but not to another.

If proteins in the native state have disulfide bonds, cysteines must be correctly oxidized. Such oxidation may be done in various ways, e.g., air oxidation, glutathione catalyzed disulfide exchange, or adduct formation followed by reduction and oxidation or by disulfide reshuffling.

Protein folding has been a topic of intensive research since Anfinsen’s demonstration that ribonu-clease can be refolded from the fully reduced and denatured state in in vitro experiments. This folding can be achieved only if the amino acid sequence itself contains all information necessary for folding into the native structure. This is the case, at least partially, for many proteins. However, a lot of other proteins do not refold in a simple one-step process. Rather, they refold via various intermediates which are relatively com-pact and possess varying degrees of secondary structures, but which lack a rigid tertiary structure. Intrachain interactions of these preformed secondary structures eventually lead to the native state. However, the absence of a rigid structure in these preformed secondary structures can also expose a cluster of hydrophobic groups to those of other polypeptide chains, rather than to their own poly-peptide segments, resulting in intermolecular aggre-gation. High efficiency in the recovery of native protein depends to a large extent on how this aggregation of intermediate forms is minimized. The use of chaperones or polyethylene glycol has been found quite effective for this purpose. The former are proteins, which aid in the proper folding of other proteins by stabilizing intermediates in the folding process and the latter serves to solvate the protein during folding and diminishes interchain aggregation events.

Protein folding is often facilitated by co-solvents, such as polyethylene glycol. As described above, proteins are functional and highly hydrated in aqueous solutions. True physiological solutions, how-ever, contain not only water but also various ions and low and high molecular weight solutes, often at very high concentrations. These ions and other solutes play a critical role in maintaining the functional structure of the proteins. When isolated from their natural environment, the protein molecules may lose these stabilizing factors and hence must be stabilized by certain compounds, often at high concentrations. These solutes are also used in vitro to assist in protein folding, and to help stabilize proteins during large-scale purification and production as well as for long-term storage. Such solutes are often called co-solvents when used at high concentrations, since at such high concentrations they also serve as a solvent along withwater molecules. These solutes encompass sugars, amino acids, inorganic and organic salts, and polyols. They may not strongly bind to proteins, but instead typically interact weakly with the protein surface to provide significant stabilizing energy without inter-fering with their functional structure.

When recombinant proteins are expressed in eukaryotic cells and secreted into media, the proteins are generally folded into the native conformation. If the proteins have sites for N-linked or O-linked glycosylation, they undergo varying degrees of glycosylation depending on the host cells used and level of expression. For many glycoproteins, glycosy-lation is not essential for folding, since they can be refolded into the native conformation without carbo-hydrates, nor is glycosylation often necessary for receptor binding and hence biological activity. However, glycosylation can alter important biological and physicochemical properties of proteins, such as pharmacokinetics, solubility, and stability.


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