Protein engineering examples

Recently Alan Fersht, Cambridge University, has developed a protein engineering procedure for such studies. The technique is based on investigation of the effects on the energetics of folding of single-site mutations in a protein of known structure. For example, if minimal mutations such as Ala to Gly in the solvent-exposed face of an a helix, destabilize both an intermediate state and the native state, as well as the transition state between them, it is likely that the helix is already fully formed in the intermediate state. If on the other hand the mutations destabilize the native state but do not affect the energy of the intermediate or transition states at all, it is likely that the helix is not formed until after the transition state. 

What can be done by predictive methods if the sequence search fails to reveal any homology with a protein of known tertiary structure Is it possible to model a tertiary structure from the amino acid sequence alone There are no methods available today to do this and obtain a model detailed enough to be of any use, for example, in drug design and protein engineering. This is, however, a very active area of research and quite promising results are being obtained in some cases it is possible to predict correctly the type of protein, a, p, or a/p, and even to derive approximations to the correct fold. 

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

Recombinant systems can also be engineered to produce receptor-mediated responses by introducing adjunct proteins. For example, it has been shown that the Gal6 G-protein subunit couples universally to nearly all... 

Enzyme promiscuity is clearly advantageous to chemists since it broadens the applicability of enzymes in chemical synthesis. New catalytic activities in existing enzymes can be enhanced by protein engineering - appropriate mutagenesis of the enzymes [106]. Some of the most illustrative examples of this unusual activity of common enzymes are presented below. 

A number of approaches may be adopted in an attempt to reduce or eliminate protein im-munogenicity. Protein engineering (Chapter 3), for example, has been employed to humanize monoclonal antibodies (Chapter 13). An alternative approach entails the covalent attachment of polyethylene glycol (PEG) to the protein backbone. This can potentially shield immunogenic epitopes upon the protein from the immune system. 

These plant defense molecules are thus of interest for a number of reasons, including their potential applications in the discovery of new pharmacological substances, their adaptation to nonproducing species, for example, for protection of crop plants from insect pests delivered either topically or via incorporation into transgenic plants, and as new structural scaffolds for protein engineering approaches. 

After the publication of the first edition of Applied Biocatalysis some five years ago, this field has rapidly been developing. This is evident from the number and types of new applications, but also from the state of the art for some of the important techniqnes, such as protein engineering and the use of non-conventional media, for example. 

Examples of fusion proteins engineered to contain a target recognition and highly potent effector... 

Bovine pancreatic RNase A is a member of a homologous superfamily. In addition, there is a separate family of guanine-specific microbial RNases that have evolved to have a similar active site.192,193 Ribonuclease T1 from Aspergillus oryzae and the 110-residue bamase from Bacillus amyloliquefaciens of Mr 12 392 (see Chapter 19) are the best known examples. One of the histidine residues is replaced by a glutamate in these enzymes. The microbial enzymes are much more amenable to study by protein engineering. 

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