Targeted random mutagenesis

List of Abbreviations GABA, gamma-aminobutyric acid type A 5HT3, 5-hydroxytryptamine type 3 SDM, site-directed mutagenesis PCR, polymerase chain reaction TRCP, targeted random chimera production SDS, sodium dodecyl sulphate... 

Fig. 2. Comparison of activities on target (p-nitrobenzyl) and screening (p-nitrophenyl) substrate of selected active pNB esterases isolated after fourth generation of random mutagenesis and screening, relative to parent enzyme from the 3rd generation. The five most active variants were pooled for random recombination...

Classical strain improvement can be used, for example, to address the regulation of an amino acid by selecting mutants that are able to grow in the presence of a mimic that has similar regulation characteristics. The classical approach involves random mutagenesis and addresses the whole genome. Hence, the phenotype is primarily targeted and the actual mechanism is not easily subjected to rational control. 

Alternatively to rational design, combinatorial approaches resembling Darwinian protein evolution through random mutagenesis, recombination, and selection for the desired properties can be employed for protein engineering. These approaches do not require extensive structural and biochemical knowledge and their implementation has resulted in successful modifications and improvements of a wide diversity of targeted properties. 

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). 

The most extensive studies on directed evolution have been carried out with Thermus aquaticus DNA Pol I (Taq Pol I). Motifs A and B have both been targeted for random mutagenesis by complementing the recA718polA12 temperature-sensitive Pol I muta-... 

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