Protein engineering

The advent of rDNA technology renders straightforward the manipulation of a protein's amino acid sequence. This process, termed site-directed mutagenesis or protein engineering, entails the controlled alteration of the nucleotide sequence coding for the polypeptide of interest such that specific, predetermined changes in amino acid sequence are introduced. Such changes can include insertions, deletions or substitutions. Site-directed mutagenesis is now most often undertaken by using a variant of the basic PCR method already described (Figure 3.15), known as 'overlap PCR', in which primers of altered nucleotide sequences are used for the PCR reactions.

Protein engineering facilitates a greater understanding of the link between a polypeptide's amino acid sequence and its structure. It also provides a powerful method of studying the relationship between structure and its function. As such, this technique will help greatly in achieving the much pursued, but still distant, objective of de novo protein design. Protein engineering is also

Table 3.3 Some approved biopharmaceuticals that have been altered by post-translational engineering

Engineered product

Insulin 'Detemir' (insulin with a fatty acid molecule attached; Chapter 11) PEGylated interferons (interferons to which polyethylene glycol molecules have been covalently attached; Chapter 8) Carbohydrate remodelled glucocerebrosidase (Cerezyme; Chapter 12)

Effect of engineering

The insulin has a prolonged duration of action

Increases product serum half-life, thereby reducing the frequency of dosage

Generation of product that is specifically targeted for uptake by macrophages used to tailor structural or functional attributes of therapeutic and other commercially important proteins.

An increasing proportion of therapeutic proteins gaining approval for general medical use display an engineered amino acid sequence, altered in order to fulfil a predefined therapeutic goal better. Selected examples of biopharmaceuticals engineered in this way are presented in Table 1.3, and these, along with additional examples, will be discussed in various subsequent chapters. An alternative approach to protein engineering entails the covalent attachment or the alteration of specific molecules/groups to or on the polypeptide's backbone (Table 3.3). Specific examples of such post-translational engineering will again be provided in subsequent chapters.

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