An alternative protein splicing mechanism for inteins lacking an N-terminal nucleophile

Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N-terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since an N-terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N-terminal splice junction (thio)ester. Despite the presence of an N-terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative protein splicing mechanism. In this non-canonical pathway, the C-extein nucleophile attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins.     

Structural and mutational studies of a hyperthermophilic intein from DNA polymerase II of Pyrococcus abyssi

Protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). Protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. We report the solution NMR structures of the hyperthermophilic Pyrococcus abyssi PolII intein, which has a noncanonical C-terminal glutamine instead of an asparagine. The NMR structures were determined to a backbone root mean square deviation of 0.46 ? and a heavy atom root mean square deviation of 0.93 ?. The Pab PolII intein has a common HINT (hedgehog intein) fold but contains an extra β-hairpin that is unique in the structures of thermophilic inteins. The NMR structures also show that the Pab PolII intein has a long and disordered loop in place of an endonuclease domain. The N-terminal Cys-1 amide is hydrogen bonded to the Thr-90 hydroxyl in the conserved block-B TXXH motif and the Cys-1 thiol forms a hydrogen bond with the block F Ser-166. Mutating Thr-90 to Ala dramatically slows N-terminal cleavage, supporting its pivotal role in promoting the N-S acyl shift. Mutagenesis also showed that Thr-90 and His-93 are synergistic in catalyzing the N-S acyl shift. The block F Ser-166 plays an important role in coordinating the steps of protein splicing. NMR spin relaxation indicates that the Pab PolII intein is significantly more rigid than mesophilic inteins, which may contribute to the higher optimal temperature for protein splicing.     

Reactivity of the cysteine residues in the protein splicing active center of the Mycobacterium tuberculosis RecA intein

Protein splicing involves the self-catalyzed excision of an intervening polypeptide segment, an intein, from a precursor protein. The first two steps in the protein splicing process lead to the formation of ester intermediates through nucleophilic attacks by the side chains of cysteine, serine, or threonine residues adjacent to the splice junctions. Since both nucleophilic residues in the Mycobacterium tuberculosis RecA intein are cysteine, their reactivities could be compared by sulfhydryl group titration. This was accomplished by using fusion proteins containing a truncated RecA intein modified by mutation to prevent protein splicing, in which the cysteines at the splice junctions were the only sulfhydryl groups. The ability to undergo hydroxylamine-induced cleavage at the upstream splice junction showed that the modified intein was not impaired in the ability to form ester intermediates. Sulfhydryl titration with iodoacetamide, monitored by quantitating the residual thiols after reaction with a maleimide derivative of biotin, revealed a striking difference in the apparent pK(a) values of the cysteines at the two splice junctions. The apparent pK(a) of the cysteine at the upstream splice junction, which initiates the N-S acyl rearrangement leading to the linear ester intermediate, was approximately 8.2, whereas that of the cysteine residue at the downstream splice junction, which initiates the transesterification reaction converting the linear ester to the branched ester intermediate, was about 5.8. This suggests that the transesterification step is facilitated by an unusually low pK(a) of the attacking thiol group. Comparison of the rates of cleavage of the linear ester intermediates derived from the M. tuberculosis RecA and the Saccharomyces cerevisiae VMA inteins by dithiothreitol and hydroxylamine revealed that the former reacted relatively more slowly with dithiothreitol, suggesting that the RecA intein has diverged in the course of evolution to react preferentially with thiolate anions and thus lacks the basic groups that may facilitate nucleophilic attack by thiols in other inteins.     

Protein splicing and auto-cleavage of bacterial intein-like domains lacking a C'-flanking nucleophilic residue

Bacterial intein-like (BIL) domains are newly identified homologs of intein protein-splicing domains. The two known types of BIL domains together with inteins and hedgehog (Hog) auto-processing domains form the Hog/intein (HINT) superfamily. BIL domains are distinct from inteins and Hogs in sequence, phylogenetic distribution, and host protein type, but little is known about their biochemical activity. Here we experimentally study the auto-processing activity of four BIL domains. An A-type BIL domain from Clostridium thermocellum showed both protein-splicing and auto-cleavage activities. The splicing is notable, because this domain has a native Ala C'-flanking residue rather than a nucleophilic residue, which is absolutely necessary for intein protein splicing. B-type BIL domains from Rhodobacter sphaeroides and Rhodobacter capsulatus cleaved their N' or C' ends. We propose an alternative protein-splicing mechanism for the A-type BIL domains. After an initial N-S acyl shift, creating a thioester bond at the N' end of the domain, the C' end of the domain is cleaved by Asn cyclization. The resulting amino end of the C'-flank attacks the thioester bond next at the N' end of the domain. This aminolysis step splices the two flanks of the domain. The B-type BIL domain cleavage activity is explained in the context of the canonical intein protein-splicing mechanism. Our results suggest that the different HINT domains have related biochemical activities of proteolytic cleavages, ligation and splicing. Yet the predominant reactions diverged in each HINT type according to their specific biological roles. We suggest that the BIL domain cleavage and splicing reactions are mechanisms for post-translationally generating protein variability, particularly in extracellular bacterial proteins.     

An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site

Protein splicing mediated by inteins is a self-processive reaction leading to the excision of the internal intein domain from a precursor protein and the concomitant ligation of the flanking sequences, the extein-N and extein-C parts, thereby reconstituting the host protein. Most inteins employ a splicing pathway in which the upstream scissile peptide bond is consecutively rearranged into two thioester or oxoester intermediates before intein excision and rearrangement into the new peptide bond occurs. The catalytically critical amino acids involved at the two splice junctions are cysteine, serine, or threonine. Notably, the only potential combination not observed so far in any of the known or engineered inteins corresponds to the transesterification from an oxoester to a thioester, which suggested that this formal uphill reaction with regard to the thermodynamic stability might be incompatible with intein-mediated catalysis. We show that corresponding mutations also led to inactive gp41-1 and AceL-TerL inteins. We report the novel GOS-TerL split intein identified from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 residues. Mutational analysis showed that its efficient splicing reaction indeed follows the shift from oxoester to thioester and thus represents a rare diversion from the canonical pathway. Furthermore, the GOS-TerL intein has an atypical split site close to the N terminus. The Int^N fragment could be shortened from 37 to 28 amino acids and exchanged with the 25-amino acid Int^N fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the Int^C fragment of the GOS-TerL intein.     

The Thermococcus kodakaraensis Tko CDC21-1 intein activates its N-terminal splice junction in the absence of a conserved histidine by a compensatory mechanism

Inteins and other self-catalytic enzymes, such as glycosylasparaginases and hedgehog precursors, initiate autocleavage by converting a peptide bond to a (thio)ester bond when Ser, Thr, or Cys undergoes an N-[S/O] acyl migration assisted by residues within the precursor. Previous studies have shown that a His at position 10 in intein Block B is essential for this initial acyl migration and N-terminal splice junction cleavage. This His is present in all inteins identified to date except the Thermococcus kodakaraensis Tko CDC21-1 intein orthologs and the inactive Arthrobacter species FB24 Arth_1007 intein. This study demonstrates that the Tko CDC21-1 intein is fully active and has replaced the lost catalytic function normally provided by the Block B His using a compensatory mechanism involving a conserved ortholog-specific basic residue (Lys(58)) present outside the standard intein conserved motifs. We propose that Lys(58) catalyzes the initial N-S acyl migration by stabilizing the thiazolidine-tetrahedral intermediate, allowing it to be resolved by water-mediated hydrolysis rather than by protonating the leaving group as His is theorized to do in many other inteins. Autoprocessing enzymes may have more flexibility in evolving catalytic variations because high reaction rates are not required when performing single-turnover reactions on "substrates" that are covalently attached to the enzyme. Consequently, inteins have more flexibility to sample catalytic mechanisms, providing insight into various strategies that enzymes use to accomplish catalysis.     

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

Inteins are the protein equivalent of introns. They are remarkable and robust single turnover enzymes that splice out of precursor proteins during post‐translational maturation of the host protein (extein). The Deinococcus radiodurans Snf2 intein is the second member of the recently discovered Class 3 subfamily of inteins to be characterized. Class 3 inteins have a unique sequence signature: (a) they start with residues other than the standard Class 1 Cys, Ser or Thr, (b) have a noncontiguous, centrally located Trp/Cys/Thr triplet, and (c) all but one have Ser or Thr at the start of the C‐extein instead of the more common Cys. We previously proposed that Class 3 inteins splice by a variation in the standard intein‐mediated protein splicing mechanism that includes a novel initiating step leading to the formation of a previously unrecognized branched intermediate. In this mechanism defined with the Class 3 prototypic Mycobacteriophage Bethlehem DnaB intein, the triplet Cys attacks the peptide bond at the N‐terminal splice junction to form the class specific branched intermediate after which the N‐extein is transferred to the side chain of the Ser, Thr, or Cys at the C‐terminal splice junction to form the standard intein branched intermediate. Analysis of the Deinococcus radiodurans Snf2 intein confirms this splicing mechanism. Moreover, the Class 3 specific Block F branched intermediate was isolated, providing the first direct proof of its existence.     

Crystal structures of an intein from the split dnaE gene of Synechocystis sp. PCC6803 reveal the catalytic model without the penultimate histidine and the mechanism of zinc ion inhibition of protein splicing

The first naturally occurring split intein was found in the dnaE gene of Synechocystis sp. PCC6803 and belongs to a subclass of inteins without a penultimate histidine residue. We describe two high-resolution crystal structures, one derived from an excised Ssp DnaE intein and the second from a splicing-deficient precursor protein. The X-ray structures indicate that His147 in the conserved block F activates the side-chain N(delta) atom of the intein C-terminal Asn159, leading to a nucleophilic attack on the peptide bond carbonyl carbon atom at the C-terminal splice site. In this process, Arg73 appears to stabilize the transition state by interacting with the carbonyl oxygen atom of the scissile bond. Arg73 also seems to substitute for the conserved penultimate histidine residue in the formation of an oxyanion hole, as previously identified in other inteins. The finding that the precursor structure contains a zinc ion chelating the highly conserved Cys160 and Asp140 reveals the structural basis of Zn2+-mediated inhibition of protein splicing. Furthermore, it is of interest to observe that the carbonyl carbon atom of Asn159 and N(eta) of Arg73 are 2.6 angstroms apart in the free intein structure and 10.6 angstroms apart in the precursor structure. The orientation change of the aromatic ring of Tyr-1 following the initial acyl shift may be a key switching event contributing to the alignment of Arg73 and the C-terminal scissile bond, and may explain the sequential reaction property of the Ssp DnaE intein.     

Splicing of the Mycobacteriophage Bethlehem DnaB Intein: IDENTIFICATION OF A NEW MECHANISTIC CLASS OF INTEINS THAT CONTAIN AN OBLIGATE BLOCK F NUCLEOPHILE*?

Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala¹ inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.     

Crystal structure of a mini-intein reveals a conserved catalytic module involved in side chain cyclization of asparagine during protein splicing

We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-A resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the N- and C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain Ndelta atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S --> O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.     

Zinc inhibition of protein trans-splicing and identification of regions essential for splicing and association of a split intein*

Two important aspects of protein splicing were investigated by employing the trans-splicing intein from the dnaE gene of Synechocystis sp. PCC6803. First, we demonstrated that both protein splicing and cleavage at the N-terminal splice junction were inhibited in the presence of zinc ion. The trans-splicing reaction was partially blocked at a concentration of 1-10 microm Zn(2+) and completely inhibited at 100 microm Zn(2+); the inhibition by zinc was reversed in the presence of ethylenediaminetetraacetic acid. We propose that inactivation of Cys(160) at the C-terminal splice junction by the chelation of zinc affects both the N-S acyl rearrangement and the transesterification steps in the splicing pathway. Furthermore, in vivo and in vitro assays were established for the determination of intein residues and regions required for splicing or association between the N- and C-terminal intein halves. N-terminal truncation of the intein C-terminal segment inhibited both splicing and association activities, suggesting this region is crucial for the formation of an interface between the two intein halves. The replacement of conserved residues in blocks B and F with alanine abolished splicing but allowed for association. This is the first evidence showing that the conserved residues in block F are required for protein splicing.     

Protein splicing in the absence of an intein penultimate histidine

Protein splicing is a self-catalytic process in which an intervening sequence, termed an intein, is excised from a protein precursor, and the flanking polypeptides are religated. The conserved intein penultimate His facilitates this reaction by assisting in Asn cyclization, which results in C-terminal splice junction cleavage. However, many inteins do not have a penultimate His. Previous splicing studies with 2 such inteins yielded contradictory results. To resolve this issue, the splicing capacity of 2 more inteins without penultimate His residues was examined. Both the Methanococcus jannaschii phosphoenolpyruvate synthase and RNA polymerase subunit A' inteins spliced. Splicing of the phosphoenolpyruvate synthase intein improved when its penultimate Phe was changed to His, but splicing of the RNA polymerase subunit A' intein was inhibited when its penultimate Gly was changed to His. We propose that inteins lacking a penultimate His (i) arose by mutation from ancestors in which a penultimate His facilitated splicing, (ii) that loss of this His inhibited, but may not have blocked, splicing, and (iii) that selective pressure for efficient expression of the RNA polymerase yielded an intein that utilizes another residue to assist Asn cyclization, changing the intein active site so that a penultimate His now inhibits splicing.     

Engineered Ssp DnaX inteins for protein splicing with flanking proline residues

Inteins are internal protein sequences capable of catalyzing a protein splicing reaction by self-excising from a precursor protein and simultaneously joining the flanking sequences with a peptide bond. Split inteins have separate pieces (N-intein and C-intein) that reassemble non-covalently to catalyze a protein trans-splicing reaction joining two polypeptides. Protein splicing has become increasingly useful tools in many fields of biological research and biotechnology. However, natural and engineered inteins have failed previously to function when being flanked by proline residue at the −1 or +2 positions, which limits general uses of inteins. In this study, different engineered inteins were tested. We found that engineered Ssp DnaX mini-intein and split inteins could carry out protein splicing with proline at the +2 positions or at both −1 and +2 positions. Under in vivo conditions in E. coli cells, the mini-intein, S1 split intein, and S11 split intein spliced efficiently, whereas the S0 split intein did not splice with proline at both −1 and +2 positions. The S1 and S11 split inteins also trans-spliced efficiently in vitro with proline at the +2 positions or at both −1 and +2 positions, but the S0 split intein trans-spliced inefficiently with proline at the +2 position and did not trans-splice with proline at both −1 and +2 positions. These findings contribute significantly to the toolbox of intein-based technologies by allowing the use of inteins in proteins having proline at the splicing point.     

Protein-splicing reaction via a thiazolidine intermediate: crystal structure of the VMA1-derived endonuclease bearing the N and C-terminal propeptides

Protein splicing excises an internal intein segment from a protein precursor precisely, and concomitantly ligates flanking N and C-extein polypeptides at the respective sides of the precursor. Here, a series of precursor recombinants bearing 11 N-extein and ten C-extein residues is prepared for the intein of the Saccharomyces cerevisiae VMA1-derived homing endonuclease referred to as VDE and as PI-SceI. The recombinant with replacements of C284S, H362N, N737S, and C738S is chosen as a spliceable precursor model and is then subjected to a 2.1A resolution crystallographic analysis. The crystal structure shows that the introduced extein polypeptides are located in the vicinity of the splicing site, and that each of their peptide bonds is in the trans conformation. The S284 O(gamma) atom located at a distance of 3.1A from the G283 C atom in the N-terminal junction suggests that a nucleophilic attack of the C284 S(gamma) atom on the G283 C atom forms a tetrahedral intermediate containing a five-membered thiazolidine ring. The tetrahedral intermediate is supposedly resolved into a thioester acyl group upon the cleavage of the linkage between the G283 C and C284 N atoms, and this thioester acyl formation completes the initial steps of Nright arrowS acyl shift at the junction between the N-extein and intein. The S738 O(gamma) atom in the C-terminal junction is placed in close proximity to the S284 O(gamma) atom at a distance of 3.6A, and is well suited for another nucleophilic attack on the resultant thioester acyl group that is then subjected to the transesterification in the next step. The reaction steps proposed for the acyl shift are driven entirely by protonation and deprotonation, in which proton ingress and egress is balanced within the splicing site.     

Compilation and analysis of intein sequences.

We have compiled a list of all the inteins (protein splicing elements) whose sequences have been published or were available from on-line sequence databases as of September 18, 1996. Analysis of the 36 available intein sequences refines the previously described intein motifs and reveals the presence of another intein motif, Block H. Furthermore, analysis of the new inteins reshapes our view of the conserved splice junction residues, since three inteins lack the intein penultimate His seen in prior examples. Comparison of intein sequences suggests that, in general, (i) inteins present in the same location within extein homologs from different organisms are very closely related to each other in paired sequence comparison or phylogenetic analysis and we suggest that they should be considered intein alleles; (ii) multiple inteins present in the same gene are no more similar to each other than to inteins present in different genes; (iii) phylogenetic analysis indicates that inteins are so divergent that trees with statistically significant branches cannot be generated except for intein alleles.     

Selection and Structure of Hyperactive Inteins: Peripheral Changes Relayed to the Catalytic Center

Inteins are phylogenetically diverse self-splicing proteins that are of great functional, evolutionary, biotechnological, and medical interest. To address the relationship between intein structure and function, particularly with respect to regulating the splicing reaction, and to groom inteins for application, we developed a phage display system to extend current in vivo selection for enhanced intein function to selection in vitro. We thereby isolated inteins that can function under excursions in temperature, pH, and denaturing environment. Remarkably, most mutations mapped to the surface of the intein, remote from the active site. We chose two mutants with enhanced splicing activity for crystallography, one of which was also subjected to NMR analysis. These studies define a “ripple effect”, whereby mutations in peripheral non-catalytic residues can cause subtle allosteric changes in the active-site environment in a way that facilitates intein activity. Altered salt-bridge formation and chemical shift changes of the mutant inteins provide a molecular rationale for their phenotypes. These fundamental insights will advance the utility of inteins in chemical biology, biotechnology, and medicine.     

Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources

Inteins excise themselves out of precursor proteins by the protein splicing reaction and have emerged as valuable protein engineering tools in numerous and diverse biotechnological applications. Split inteins have recently attracted particular interest because of the opportunities associated with generating a protein from two separate polypeptides and with trans-cleavage applications made possible by split intein mutants. However, natural split inteins are rare and differ greatly in their usefulness with regard to the achievable rates and yields. Here we report the first functional characterization of new split inteins previously identified by bioinformatics from metagenomic sources. The N- and C-terminal fragments of the four inteins gp41-1, gp41-8, NrdJ-1, and IMPDH-1 were prepared as fusion constructs with model proteins. Upon incubation of complementary pairs, we observed trans-splicing reactions with unprecedented rates and yields for all four inteins. Furthermore, no side reactions were detectable, and the precursor constructs were consumed virtually quantitatively. The rate for the gp41-1 intein, the most active intein on all accounts, was k = 1.8 ± 0.5 × 10(-1) s(-1), which is ～10-fold faster than the rate reported for the Npu DnaE intein and gives rise to completed reactions within 20-30 s. No cross-reactivity in exogenous combinations was observed. Using C1A mutants, all inteins were efficient in the C-terminal cleavage reaction, albeit at lower rates. C-terminal cleavage could be performed under a wide range of reaction conditions and also in the absence of native extein residues flanking the intein. Thus, these inteins hold great potential for splicing and cleavage applications.     

Intein-mediated ligation and cyclization of expressed proteins

Protein splicing is a posttranslational processing event that releases an internal protein sequence from a protein precursor. During the splicing process the internal protein sequence, termed an intein, embedded in the protein precursor self-catalyzes its excision and the ligation of the flanking protein regions, termed exteins. The dissection of the splicing pathway, which involves the precise cleavage and formation of peptide bonds, and the identification of key catalytic residues at the splice junctions have led to the modulation of the protein splicing process as a protein engineering tool. Novel strategies have been developed to use intein-catalyzed reactions for the production and manipulation of proteins and peptides. These new approaches have broken down the size limitation barrier of chemical synthetic methods and are less technically demanding. The purpose of this article is to describe how to use self-splicing inteins in protein semisynthesis and backbone cyclization. The first two sections of the article provide a brief review of the distinct chemical steps that underlie protein splicing and intein enabled technology.     

蛋白质剪切及其应用

蛋白质剪切是一种翻译后修饰事件 ,它将插入前体蛋白的中间的蛋白质肽段 (Intein ,internalproteinfrag ment)剪切出来 ,并用正常肽键将两侧蛋白质多肽链 (Extein ,flankingproteinfragments)连接起来。在此过程中不需要辅酶或辅助因子的作用 ,仅需四步分子内反应。Intein及其侧翼序列可以通过突变产生高度特异性的自我切割用于蛋白质纯化、蛋白质连接和蛋白质环化反应 ,在蛋白质工程方面有广泛的应用前景。     

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

Protein splicing catalyzed by inteins has enabled various biotechnological applications such as protein ligation. Successful applications of inteins are often limited by splicing efficiency. Here, we report the comparison of protein splicing between 20 different inteins from various organisms in identical contexts to identify robust inteins with foreign exteins. We found that RadA intein from Pyrococcus horikoshii and an engineered DnaB intein from Nostoc punctiforme demonstrated an equally efficient splicing activity to the previously reported highly efficient DnaE intein from Nostoc punctiforme. The newly identified inteins with efficient cis-splicing activity can be good starting points for the further development of new protein engineering tools.