Exploration of the origin and evolution of globular proteins by mRNA display.

The questions of how proteins first appeared on the primitive earth and how they evolved into functional proteins are fundamental. If we can understand the origins and evolution of proteins, we should be able to create novel functional proteins. Evolutionary protein engineering or directed protein evolution has been used to create artificial proteins with novel functions by repeated mutation, selection, and amplification, mimicking Darwinian evolution in the laboratory. For this purpose, display technology, such as mRNA display, to link genotype with phenotype is extremely important. Here I focus on three hypotheses regarding the origin and evolution of proteins. First, Eigen's GNC hypothesis proposes that the early genetic code began from the directionless codons GNC and GNN, where N denotes U, C, A, or G. Second, Ohno's gene duplication theory proposes that gene duplication produces two functionally redundant, paralogous genes, of which one retains the original function, leaving the second free to evolve adaptively. Third, Gilbert's exon shuffling theory proposes that new genes are formed through shuffling of small segments corresponding to exons. I then review various experimental approaches to evolutionary protein engineering using mRNA display, such as the creation of functional proteins from random sequences with limited sets of amino acids, randomly mutated folded proteins, and block-shuffled sequence proteins, and I discuss the results in relation to these three hypotheses.