Fig. 9.2 Scheme of several combinatorial tools for selections. A physical entity ensures the link between phenotype (protein = blue) and genotype (gene = green). It can be phages, cells, ribosomes or puromycin molecules for example. These systems allow firstly the isolation of proteins according to their function from a library of proteins and secondly the characterization of selected proteins by amplification and sequencing of the corresponding genes linked to it.

Selections are generally done by binding to a ligand immobilized on a solid surface to facilitate washings. After each round of selection, the binders of interest are amplified via cell multiplication, phage propagation, or PCR depending on the method of selection used. Finally, after several rounds of selection, the selected variants are isolated, analyzed for the desired properties, and their sequence identified.

After several decades of protein folding studies, it is still a real challenge to design fully de novo proteins. Using rules deduced from sequence-structure-function relationships for natural proteins, some proteins with simple folds, such as a four helix bundle fold, have been successfully designed de novo.

In this chapter key challenges in protein engineering will be reviewed focusing on the strategies used to isolate a protein with the desired properties. Strategies that make use of natural scaffolds, of domains, of secondary structures, of altered amino acid alphabets and strategies that do not make use of known protein sequences will be addressed here.

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