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Protein engineering using ancestral reconstruction techniques

Most people associate ancestors with their great-grandparents and family lineage. However, and not surprisingly, given the nature of evolution, the molecular ancestors of our proteins often reside in other species.
Protein engineering using ancestral reconstruction techniques
Advances in molecular evolution have enabled exploration of the mechanisms by which protein families diverge. The search is on for experimental techniques leading to ancestral resurrections or reconstructions. Experiments to date have shown that reconstructed ancestors are more susceptible to functional divergence. They are more stable across a wide temperature range, with possibly higher likelihood of evolving than other protein families. Although the potential for developing genetically engineered mutants with novel functionalities is high, existing techniques are too limited in scope to make this a reality.

Scientists initiated the EU-funded project 'Protein archaeology: Reconstructed ancestors for protein engineering and crystallography' (PROTEINARCHEOLOGY) to develop methodologies with better capabilities. Ancestral resurrection techniques developed within the scope of the project were applied to two protein families to isolate active clones with superior catalytic activity. This technique required screening of only 200–300 variants as opposed to the thousands required with classical directed evolution experiments. Thus, ancestral libraries provide a powerful starting point for enzyme engineering to improve catalytic functionality in generated variants with increased stability and solubility.

Further, scientists applied the techniques to gain greater understanding of the evolutionary mechanisms of phosphate-binding proteins (PBPs). Phosphate binding is a function seen in ancient organisms. PBPs exhibit great affinity for phosphate and are critical to the fundamental cellular process of phosphate uptake. Researchers undertook the detailed biochemical and structural characterisation of five PBPs. Results demonstrated that this family is under selection pressure for their high phosphate affinity and their extremely high selectivity. Groundbreaking work demonstrated the mechanism of this unprecedented selectivity. This provides options for development of ultra-selective drug therapies and numerous other industrially relevant, highly specific molecules.

PROTEINARCHEOLOGY has opened the door to the highly efficient engineering of proteins having broad sweeping implications for industrial chemistry and therapeutics. The powerful tools will also be essential to unravelling the mechanisms of molecular evolution and factors that drive evolution.

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