Identification of functional Npu DnaE and gp41-1 inteins split in three fragments

Inteins are special proteins that auto-catalytically carry out a protein splicing reaction. Due to their ability to post-translationally modify target proteins in vitro and in vivo, they are used in different applications, ranging from protein purification to the construction of Boolean logic gates. So far inteins have been found to be either encoded by a single gene (contiguous inteins) or by two separate ones (split inteins). Previously, it has been shown that the contiguous Ssp and Rma DnaB inteins could be artificially split in three fragments and retain functionality. Here we report the identification of novel split sites within the N-terminal fragments of the ultra-fast and efficient Npu DnaE and gp41-1 split inteins that lead to synthetic functional three-piece versions of these inteins. Our data suggest that splitting inteins in three fragments is generally applicable and pave the way for new biotechnological applications based on highly-fragmented inteins.