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Chapter: Biotechnology Applying the Genetic Revolution: Recombinant Proteins

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Increasing Protein Stability

Different proteins vary greatly in stability. The lifetime of a protein inside a cell depends mainly on how fast the protein is degraded by proteolytic enzymes or proteases.

INCREASING PROTEIN STABILITY

Different proteins vary greatly in stability. The lifetime of a protein inside a cell depends mainly on how fast the protein is degraded by proteolytic enzymes or proteases. Apart from the overall 3D structure of the protein, there are two specific factors that greatly affect the rate of protein degradation. These are believed to act by altering the recognition of proteins by the degradation machinery.

 

(a)   The identity of the N-terminal amino acid greatly affects the half-life of proteins. Although all polypeptide chains are made with methionine as the initial amino acid, many proteins are processed later. Therefore the N-terminal amino acid of mature proteins varies greatly. The N-terminal rule for stability is shown in Table 10.2.

 

(b)  The presence of certain internal sequences greatly destabilizes proteins. PEST sequences are regions of 10 to 60 amino acids that are rich in P (proline), E (glutamate), S (serine), and T (threonine). The PEST sequences create domains whose structures are recognized by proteolytic enzymes.



Alteration of the N-terminal sequence of a protein by genetic engineering is relatively simple. A small segment of artificially synthesized DNA encoding a new N-terminal region can be incorporated by standard methods, e.g., PCR (see Chapter 4). The effectiveness of this approach has been demonstrated experimentally for β-galactosidase. Changing an internal PEST sequence without disrupting protein function is a vastly more complex undertaking, even though in theory it could lead to greater stability and therefore greater yields.


In some instances, proteins, which would be stable if folded correctly, fold aberrantly during synthesis. The misfolded proteins form inclusion bodies, which are dense particles visible with optical microscopy. Inclusion bodies are usually formed when recombinant proteins are poorly soluble or produced too fast. One method to alleviate the aggregation is to provide molecular chaperones that help fold the recombinant protein correctly. Molecular chaperones attach themselves to polypeptides while they are being translated and help keep the protein unfolded until translation is complete. Then the protein can fold into its correct shape (Fig. 10.6).


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