Directed protein sequence space

These initial systematic studies regarding the directed evolution of PAL allowed several conclusions to be made. Protein sequence space can be explored successfully by applying the following strategies [8c,33j ... 

Reetz MT, Wang LW, Bocola M (2006) Directed evolution of enantioselective enzymes iterative cycles of CASTing for probing protein-sequence space. Angew Chem Int Ed 45 1236-1241... 

Subsequent directed evolution work on Pseudomonas aeruginosa demonstrates that protein sequence space with respect to enantioselectivity is best explored by a three-step procedure (Reetz, 2001) (i) generation of mutants by error-prone PCR at a high-mutation rate (ii) identification of hot regions and spots in the enzyme by error-prone PCR and substantiation by simplified combinatorial multiple-cassette mutagenesis (iii) extension of the process of combinatorial multiple-cassette mutagenesis to cover a defined region of protein sequence space. 

The superior performance of proteins in respect to functional diversity, specificity, and efficiency has attracted scientists and engineers alike to create tailor-made catalysts by protein engineering. Customizing a protein towards practical applications such as industrial catalysts, biosensors, and therapeutics offers many economic incentives. At the same time, protein engineering can address fundamental questions of protein design in respect to their structure and function. This can then lead to the development of new technologies facilitating the more directed exploration of protein sequence space. 

Fig. 8. Amino acid sequences of mature dinoflagellate iPCP proteins. Sequence a is a consensus sequence obtained by direct protein sequencing, b,c and d by RT-PCR and e and f from genomic sequencing (Hiller et al.,1995 and R. G.Hiller and P.M. Wrench, unpublished). a space identity.

Functional tolerance is a significant factor for the success of directed evoluiton. A protein that is functionally tolerant allows many mutations without disrupting the fitness, making it more likely that there is a connected path in sequence space of single mutations that leads to regions of higher fitness. Tolerance also affects the quality of the mutant library. If the protein is functionally intolerant, the mutant library will consist of mostly inactive proteins. 

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