Evolution has lead to a wide variety of proteins, which together enable the existence of all life. The proteins known, and predicted, to exist on earth are a minute fraction of the potential protein universe. Nature has not sampled all the possible amino acid sequences, and therefore, in all likelihood, it has not produced all viable protein folds. Instead, nature has evolved a protein repertoire which is nothing more than sufficient. This project aims to explore whether new folds can be produced from the same building blocks used to produce natural proteins. These building blocks are sub-domains. Sub-domains are small fragments of proteins (15-80 amino acids) which contribute some selectable protein feature. It has been proposed that transient association of sub-domains led to the formation of oligomers resembling modern domains. The micro genes encoding the oligomeric sub-domains are thought to have fused, leading to modern genes encoding modern protein domains. We are trying to shuffle sub-domains from within modern proteins in vitro using the method Incremental Truncation for the Creation of Hybrid enzYmes (ITCHY), which combines fragments from any two non-homologous genes. We have implemented ITCHY with the pooled clones of the ASKA collection (~4,000 plasmids for the over-expression of almost every E. coli open reading frame), generating a library of hybrids that are made up of random fragments from any two E. coli genes. A system for the genetic selection of folded proteins has been used to identify candidates that may possess novel folds. Functional screening indicated that some of these chimeric proteins may confer growth advantages on E. coli under specific environmental conditions. Together these data suggest that sub-domain shuffling can lead to the generation of new proteins, with structures and potentially functions that are different from the parental proteins.

Evolving new proteins by non-homologous recombination

254th National Meeting (2017)

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