Abstract
Inteins are prevalent among extremophiles. Mini-inteins with robust splicing properties are of particular interest for biotechnological applications due to their small size. However, biochemical and structural characterization has still been limited to a small number of inteins, and only a few serve as widely used tools in protein engineering. We determined the crystal structure of a naturally occurring Pol-II mini-intein from Pyrococcus horikoshii and compared all three mini-inteins found in the genome of P. horikoshii. Despite their similar sizes, the comparison revealed distinct differences in the insertions and deletions, implying specific evolutionary pathways from distinct ancestral origins. Our studies suggest that sporadically distributed mini-inteins might be more promising for further protein engineering applications than highly conserved mini-inteins. Structural investigations of additional inteins could guide the shortest path to finding novel robust mini-inteins suitable for various protein engineering purposes.
Highlights
Self-splicing protein introns are genetic elements that are translated with their host proteins [1,2]
Biochemical as well as structural studies of diversely or arbitrarily selected inteins were performed and resulted in serendipitously identifying robust inteins, which could be suitable for protein engineering [15,19]
There is a very limited number of inteins suitable for biotechnological applications because such inteins require (i) a most robust splicing activity in vivo and in vitro, (ii) fast reaction kinetics, (iii) a high tolerance of foreign extein contexts, and (iv) functional reconstruction of their catalytically active structures from split fragments in order to enable a versatile use of protein splicing [10,14,15]
Summary
Self-splicing protein introns (inteins) are genetic elements that are translated with their host proteins [1,2]. Horizontal gene transfers (HGT) that occurred during evolution contributed to this variation, counterbalanced by the degeneration events of the nested homing endonuclease domains (HENs) [3] Due to these evolutionary events, the structure and protein-splicing activities of inteins might represent the evolutionary history of each intein [9]. As the first step towards a rational approach to identify robust inteins for protein engineering, we turned our attention to the 14 inteins identified in the genome of P. horikoshii. Three of these inteins can be classified as mini-inteins (≤200 residues). The structural comparison and accumulated biochemical data might serve as a practical compass in the quest for robust mini-inteins from genomic sequence data
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