Inteins, valuable genetic elements in molecular biology and biotechnology

Abstract

Inteins are internal protein elements that self-excise from their host protein and catalyze ligation of the flanking sequences (exteins) with a peptide bond. They are found in organisms in all three domains of life, and in viral proteins. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. This self-excision process is called protein splicing, by analogy to the splicing of RNA introns from pre-mRNA. Protein splicing involves only four intramolecular reactions, and a small number of key catalytic residues in the intein and exteins. Protein-splicing can also occur in trans. In this case, the intein is separated into N- and C-terminal domains, which are synthesized as separate components, each joined to an extein. The intein domains reassemble and link the joined exteins into a single functional protein. Understanding the cis- and trans-protein splicing mechanisms led to the development of intein-mediated protein-engineering applications, such as protein purification, ligation, cyclization, and selenoprotein production. This review summarizes the catalytic activities and structures of inteins, and focuses on the advantages of some recent intein applications in molecular biology and biotechnology.

Fig. 1

Protein splicing. The intein coding sequence is transcribed into mRNA and translated to a nonfunctional protein precursor, which then undergoes a self-catalyzed rearrangement in which the intein is excised and the exteins are joined to yield the mature protein

Fig. 2

Structure of large and mini-inteins. Conserved elements in a large intein and mini-intein are indicated. The white and grey areas A, N2, B, N4, C, D, E, H, F, and G are conserved intein motifs identified by Pietrokovski (1994, 1998) and Perler et al. (1994). The exteins are illustrated in black and the intein sequence in blue. The site of insertion of the homing endonuclease in large inteins is indicated by the dark vertical line. Conserved amino acid residues of the intein and the C-extein are indicated below

Fig. 3

Splicing mechanism of inteins. Intein splicing takes place in four reaction steps. See text for details

Fig. 4

Intein-mediated protein purification. Expression and purification of a protein fused to the N-terminus of a mutated intein variant. The intein is further fused to a chitin binding domain (CBD) at its C-terminus. Purification of the fusion construct is accomplished by the utilization of a chitin column. The intein is mutated at the C-terminus (Asnⁿ), so that cleavage only occurs at the N-terminus, which results in the release of the target protein from the column while the intein-CBD tag remains bound to chitin

Fig. 5

Self-circularization of peptides. a Self-circularization of proteins using the TWIN-system. A target protein is embedded between two intein sequences, which are modified for N- or C-terminal cleavage, respectively. The inducible splicing reaction of the inteins leads to the generation of an activated thioester residue and an N-terminal Cys for the spontaneous circularization of the linear peptide. b Utilization of the Split Intein-mediated Ciruclar Ligation Of Peptides a ProteinS (SICLOPPS) also enables the circularization of peptides. In this system, the order of the naturally split Synechocystis sp. Ssp DnaE intein is inverted (I_C–target protein–I_N), and the reconstitution of the Ssp DnaE intein allows the efficient cyclization of the target protein

Fig. 6

Intein-mediated production of Sec-containing proteins. A UGA-codon is translated to a Sec, when a SECIS element is in close proximity at the N-terminal part of the modified fungal Pch PRP8-intein. The incorporation of the Sec residue followed by the protein splicing process leads to the production of a mature Sec-containing protein