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Because present-day proteins are composed of 20 kinds of amino acids, the number of possible amino-acid sequences in a 100-residue protein is 20100 (approximately 10130), which is larger than the total number of atoms in the universe (~1080). The number of proteins that may have existed in nature throughout the history of life on the Earth has been estimated to be less than 1050 molecules (Mandecki, 1998) or 1043 molecules (Dryden et al., 2008). Thus, the vast sequence space available remains to be explored further, and the sequence space that remains unexplored provides an opportunity to create valuable proteins with novel structures and functions for biomedical and environmental applications. Evolutionary protein engineering or directed protein evolution has been used to create artificial proteins with novel functions (Bloom et al., 2005; Hoogenboom, 2005; Leemhuis et al., 2005; Romero & Arnold, 2009) by repeated mutation, selection and amplification, mimicking Darwinian evolution in the laboratory (Figure 1).

Evolutionary Engineering of Artificial Proteins with Limited Sets of Primitive Amino Acids