Inteins: Localized Distribution, Gene Regulation, and Protein Engineering for Biological Applications

Abstract

Inteins are self-splicing polypeptides with an ability to excise themselves from flanking host protein regions with remarkable precision; in the process, they ligate flanked host protein fragments. Inteins are distributed sporadically across all three domains of life (bacteria, archaea, and unicellular eukaryotes). However, their apparent localized distribution in DNA replication, repair, and recombination proteins (the 3Rs), particularly in bacteria and archaea, is enigmatic. Our understanding of the localized distribution of inteins in the 3Rs, and their possible regulatory role in such distribution, is still only partial. Nevertheless, understanding the chemistry of post-translational self-splicing of inteins has opened up opportunities for protein chemists to modify, manipulate, and bioengineer proteins. Protein-splicing technology is adapted to a wide range of applications, starting with untagged protein purification, site-specific protein labeling, protein biotinylation, isotope incorporation, peptide cyclization, as an antimicrobial target, and so on. This review is focused on the chemistry of splicing; the localized distribution of inteins, particularly in the 3Rs and their possible role in regulating host protein function; and finally, the use of protein-splicing technology in various protein engineering applications.

Inteins in Science: Evolution to Application

“…Since then, further examples of inteins were found in all three domains of life—in archaea, the DNA polymerase of the extremely thermophilic archaebacteria Thermococcus litoralis [ 36 ], in bacteria, the RecA proteins of M. tuberculosis [ 37 , 38 ] and M. leprae [ 39 ] and in eukarya, the 69 kDa subunit of vacuolar ATPase of the yeast Candida tropicalis [ 40 ]. This highlights a wider distribution of inteins across all three domains of life ( Figure 2 b), suggesting an ancient origin that predates the separation of prokaryotes and eukaryotes [ 18 , 20 , 41 ]. We dug into the NCBI Gene data base ( ) to scan the distribution of intein in all the three domains of life, where out of 2709 intein-containing genomes, 56% of total intein-containing genome is found in eukaryotes, 19.8% in archaea and 6.64% in eubacteria.…”

“…The two fragments reassemble into a complete intein structure that functions similar to a full-length intein system [ 50 , 54 , 55 ]. When inteins express themselves as a contiguous system, such as in a full-length intein, it is known as cis -splicing [ 20 , 56 ]. The split-intein system, expressed as two separate genes follow a trans -splicing system instead.…”

“…The study of intein distribution, dissemination and their potential biological functions are particularly fascinating in the field of translational research. Inteins distribution is sporadic in the genomes of organisms spanning from archaea, bacteria and eukaryotes to several viral genomes [ 18 , 19 , 20 ]. The reason for such anomalous distribution has spurred the proposal for numerous evolutionary scenarios, including the role of inteins in genetic mobility and as a selfish DNA [ 19 , 20 , 21 ].…”

Evolving a Peptide: Library Platforms and Diversification Strategies

“…It takes advantage of intein splicing to generate peptide libraries in cells [140]. Inteins are self-excising protein domains that process to link their consecutive sequences with a peptide bond, while liberating the intervening portions (exteins) as head-to-tail cyclized peptides [141]. Library peptides to be cyclized are usually 6 amino acids long randomized exteins, flanked by C-and N-terminal intein domains with splice site-adjacent cysteine residues.…”

The outer-membrane protein MafA of Neisseria meningitidis constitutes a novel protein secretion pathway specific for the fratricide protein MafB

“…DUF1020, and a C-terminal toxic domain. In some MafB proteins, a Hint domain, which is expected to mediate protein self-splicing [ 12 , 13 ] separates the DUF1020 from the toxic domain. The mafB genes are localized on the chromosome in genomic islands designated Maf Genomic Islands (MGI).…”