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Split inteins are biological mechanisms for the operation of the spatiotemporal control of protein activities. They function through protein trans -splicing, in which their N- and C-terminal fragments are expressed contiguously with two protein halves. The subsequent self-excision upon recognition of the complimentary fragment yields a mature, complete, and functional protein. The conditional regulation of protein splicing through environmental factors or the attachment of regulatory modules can be used to determine when and where a protein will operate, providing potential novel approaches for engineering biology applications. This review will discuss current split intein applications and the mechanistic basis for novel species classification. These considerations can provide guidance in intein and extein engineering through activation strategies, in the design of spatial arrangements, and in taking advantage of unique reaction environments. This can pave the way for the future implementation of novel split intein discoveries and the selection of appropriate intein species and aid in designing novel protein engineering strategies.

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)