Directed evolution of protein function

One of the simplest approaches to molecular breeding is to begin with a gene for an enzyme that performs a certain activity, then introduce variation or molecular diversity at the genetic level into the gene by a number of methods including chemical mutagenesis, error-prone PGR, 

Wiidtype recombinant singie stranded tempiate piasmid DNA [ (-) strand DNA] 

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All these approaches have been used to alter protein function, to increase the activity or solubility of proteins, or to adapt enzymes for industrial applications. The goal of artificial man-made proteins with tailor-made activities is, however, still far away and none of the currently existing approaches provides the ultimate solution to the directed evolution of proteins. Nevertheless, numerous examples of successfully altered and improved proteins clearly show the power of directed evolution for protein design. 

The directed evolution of proteins by random mutagenesis and DNA shuffling has proven extremely powerful to modify and optimize functional and physicochemical properties of existing proteins [6, 79 - 85] (for an up-dated list on targets see http //www.che.caltech.edu / groups / fha/Enzyme/directed.html). 

Yan, X. and Xu, Z. (2006) Ribosome-display technology applications for directed evolution of functional proteins. Drug Discov Today 11, 911-916. 

In contrast to rational approaches, the directed evolution of enzymes is based on the search of useful functionalities in libraries randomly generated and on improvement by suitable and proper selection. The directed evolution combines two powerful and independent technologies methods for the generation of random genetic libraries and strategies for the selection of variant enzymes with the specific capabilities [499-503]. This process can result in biocatalysts with non-natural proprieties, since the proteins are expressed in recombinant cells decoupled from its biological functions and evolved under unusual conditions. One additional advantage is the possibility to tailor not only individual proteins, but also the whole biosynthetic and catabohc pathways [471]. 

Although directed evolution is a powerful tool for protein design, it is limited by the number of protein variants that can be screened experimentally. As shown in Table 1, for a protein of typical size (300 amino acids), the library size of protein variants with each variant containing three mutations is over 3 xlO, which is too large to be examined experimentally. However, it is possible that even more simultaneous mutations are needed in a protein to achieve novel protein activity or drastic improvement of protein functions. On the other hand, although rational design has been used for more than two decades, our... 

Leemhuis, H. et al (2005) New genotype—phenotype linkages for directed evolution of functional proteins. Curr. Opin. Struct. Biol, 15 (4), 472 -478. 

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