Directed Molecular Evolution of Proteins

Petra Tafelmeyer, and KaiJohnsson

Directed molecular evolution adopts the Darwinian approach to the evolution of proteins or peptides and, in contrast to rational approaches, does not require information about the sequence and the structure of the protein. In short, directed evolution consists in repetitive cycles of random mutagenesis of the protein/peptide sequence followed by screening or selection for candidates with the desired properties (Figure B.20.1).

Many different approaches are used to introduce mutations into a gene; most of those currently used are based on the polymerase chain reaction (PCR). Error-prone PCR, for example, uses a low-fidelity DNA polymerase and reaction conditions likely to introduce random base changes into the polynucleotide chain [1]. Care must be taken regarding the mutation rate -although a low level of mutagenesis will cover only a small fraction of the accessible sequence space, higher mutation rates also increase the chances of accumulating unfavorable or deleterious mutations, resulting in non-active protein. A combinatorial approach for enzyme evolution, called DNA shuffling, consists in random fragmentation of a gene then reassembly by self-priming PCR; this enables recombination of mutations from different

Fig. B.20.1. Example of the directed molecular evolution of a protein.

clones [2]. To further increase the accessible sequence space, DNA shuffling even enables recombination of families of homologous genes to generate protein chimeras [3].

In contrast with error-prone PCR and DNA shuffling, saturation mutagenesis enables randomization of a particular residue (or several residues) in the protein of interest with any of the 20 natural amino acids [4]. In this process, however, additional information is required to determine interesting positions for randomization.

More critical than choosing a particular method for randomization is the choice of a suitable screening or selection scheme to identify the clones with the desired activity from a library of an immense number of inactive or less active mutants (current library sizes vary between 104 and 1013 individual members). In other words, a link between the desired property (i.e. phenotype) and the corresponding gene (i.e. genotype) must be created.

In general, one must distinguish between screening and selection methods. Screening relies on inspection of all individual members of a library and isolation of the interesting ones on the basis of specific properties of the active mutants (often by visual or spectroscopic detection). Selection, in contrast, is based on elimination of undesired variants. This can be achieved either on the basis of the significant growth advantage provided by the active protein to its host or, in vitro, as a result of ligand binding and temporary immobilization of the active variants while undesired mutants are washed away. Techniques used in screening or selection experiments range from facile colony activity screening to yeast two-hybrid systems or in-vitro selection display systems (phage display, mRNA display, ribosome display), to mention only some of the numerous possibilities [5-10].

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.

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