Synthetic Two-piece and Three-piece Split Inteins for Protein trans-Splicing

Abstract

Inteins are protein-intervening sequences that can self-excise and concomitantly splice together the flanking polypeptides. Two-piece split inteins capable of protein trans-splicing have been found in nature and engineered in laboratories, but they all have a similar split site corresponding to the endonuclease domain of the intein. Can inteins be split at other sites and do transsplicing? After testing 13 split sites engineered into a Ssp DnaB mini-intein, we report the finding of three new split sites that each produced a two-piece split intein capable of protein trans-splicing. These three functional split sites are located in different loop regions between ␤-strands of the intein structure, and one of them is just 11 amino acids from the beginning of the intein. Because different inteins have similar structures and similar ␤-strands, these new split sites may be generalized to other inteins. We have also demonstrated for the first time that a three-piece split intein could function in protein trans-splicing. These findings have implications for intein structure-function, evolution, and uses in biotechnology.An intein is a protein-intervening sequence that catalyzes a protein-splicing reaction in which the intein sequence is precisely excised and its flanking sequences (N-and C-exteins) join with a peptide bond to produce the mature host protein (spliced protein) (1). The mechanism of protein splicing typically has four steps: two acyl rearrangements at the two splicing junctions, a trans-esterification between the two junctions, and a cyclization of the Asn residue at the C-terminal junction (2-4). Crystal structures of inteins revealed a splicing domain consisting of 11-12 ␤-strands and forming a compact horseshoe shape with the splicing junctions located in the central cleft (5-11). A majority of inteins also have a homing endonuclease domain inserted in the splicing domain sequence (12). These bifunctional inteins are ϳ350 -550 amino acids (aa) 1 long, although some extra large inteins are up to 1650 aa long and also contain tandem repeats (13,14). Nearly 200 intein and inteinlike sequences have been found in a wide variety of host proteins and in microorganisms belonging to bacteria, Archaea, and eukaryotes (12,15). Their sporadic phylogenetic distributions suggest lateral gene transfer through intein homing (16,17). Inteins generally share only low levels of sequence similarity, but they share striking similarities in structure, reaction mechanism, and evolution (4,18,19,21). It is thought that inteins first originated with just the splicing domain and then acquired the endonuclease domain, with the latter conferring genetic mobility to the intein. During intein evolution, however, some inteins lost their endonuclease domain to become mini-inteins consisting of just the ϳ130-aa protein-splicing domain plus a linker sequence of various lengths in place of the endonuclease domain (12,22).An interesting event of intein evolution is the loss of sequence continuity in some inteins, which apparently produced ...

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

“…pH Urea gp41-1 (1 ؉ 6) C-extein sequence (proteins [15][16][17][18] and tested for C-cleavage activity when the purified proteins were mixed with the complementary Int N C1A fusions (proteins [11][12][13][14]. In Fig.…”

“…1A, the translation of both fragments reconstitutes the catalytic activity of protein trans-splicing to form the mature protein (12). Split inteins can also be generated artificially by dividing inteins into two or three fragments (13). In the case of the Ssp DnaB intein, the N-terminal intein fragment (Int N ) 3 could be as short as 11 amino acids (14), whereas 6 amino acids were sufficient for the Int C fragment of the Ssp GyrB intein (15).…”

Evaluation and comparison of protein splicing by exogenous inteins with foreign exteins inEscherichia coli

“…Other primer sequences are listed in Supporting Information (Table S1). The 13 genes for (1)- (9), (12), (14), (16), and (20) in Table 1 were obtained by PCR from genomic DNAs (ATCC) or commercial plasmids (New England Biolabs) using the listed oligonucleotides. The first residue of (1) Mja KlbA intein in pSKDuet19, which is mutated to serine, was changed back to alanine by QuikChange Ò protocol (Stratagene) using the two oligonucleotides HK401 and HK402, resulting in pSEDuet33.…”

Interaction Studies and Alanine Scanning Analysis of a Semi-synthetic Split Intein Reveal Thiazoline Ring Formation from an Intermediate of the Protein Splicing Reaction

“…Other more recently emerging exciting avenues for protein engineering approaches rely on protein trans-splicing. This reaction is performed by split inteins, which are found in a few native examples (10,11) or can be created artificially from a regular intein (12)(13)(14)(15)(16)(17)(18)(19). Protein trans-splicing enables the post-translational linkage of proteins or polypeptides originating from two separate molecules.…”