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Intein-mediated protein splicing raises questions and creates opportunities in many scientific areas. Evolutionary biologists question whether inteins are primordial enzymes or simply selfish elements, whereas biochemists seek to understand how inteins work. Synthetic chemists exploit inteins in the semisynthesis of proteins with or without nonribosomal modifications, whereas biotechnologists use modified inteins in an ever increasing variety of applications. The four minireviews in this series explore these themes. The first minireview focuses on the evolution and biological function of inteins, whereas the second describes the mechanisms that underlie the remarkable ability of inteins to perform complex sets of choreographed enzymatic reactions. The third explores the relationship between the three-dimensional structure and dynamics of inteins and their biochemical capabilities. The fourth describes intein applications that have moved beyond simple technology development to utilizing inteins in more sophisticated applications, such as biosensors for identifying ligands of human hormone receptors or improved methods of biofuel and plant-based sugar production., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Inteins are nature's escape artists; they facilitate their excision from flanking polypeptides (exteins) concomitant with extein ligation to produce a mature host protein. Splicing requires sequential nucleophilic displacement reactions catalyzed by strategies similar to proteases and asparagine lyases. Inteins require precise reaction coordination rather than rapid turnover or tight substrate binding because they are single turnover enzymes with covalently linked substrates. This has allowed inteins to explore alternative mechanisms with different steps or to use different methods for activation and coordination of the steps. Pressing issues include understanding the underlying details of catalysis and how the splicing steps are controlled., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The hyperthermophilic archaeon Thermococcus litoralis strain NS-C, first isolated in 1985, has been a foundational organism for archaeal research in biocatalysis, DNA replication, metabolism, and the discovery of inteins. Here, we present the genome sequence of T. litoralis with a focus on the replication machinery and inteins.

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.

We report the first detailed investigation of the kinetics of protein splicing by the Methanococcus jannaschii KlbA (Mja KlbA) intein. This intein has an N-terminal Ala in place of the nucleophilic Cys or Ser residue that normally initiates splicing but nevertheless splices efficiently in vivo [Southworth, M. W., Benner, J., and Perler, F. B. (2000) EMBO J.19, 5019-5026]. To date, the spontaneous nature of the cis splicing reaction has hindered its examination in vitro. For this reason, we constructed an Mja KlbA intein-mini-extein precursor using intein-mediated protein ligation and engineered a disulfide redox switch that permits initiation of the splicing reaction by the addition of a reducing agent such as dithiothreitol (DTT). A fluorescent tag at the C-terminus of the C-extein permits monitoring of the progress of the reaction. Kinetic analysis of the splicing reaction of the wild-type precursor (with no substitutions in known nucleophiles or assisting groups) at various DTT concentrations shows that formation of the branched intermediate from the precursor is reversible (forward rate constant of 1.5 × 10(-3) s(-1) and reverse rate constant of 1.7 × 10(-5) s(-1) at 42 °C), whereas the productive decay of this intermediate to form the ligated exteins is faster and occurs with a rate constant of 2.2 × 10(-3) s(-1). This finding conflicts with reports about standard inteins, for which Asn cyclization has been assigned as the rate-determining step of the splicing reaction. Despite being the slowest step of the reaction, branched intermediate formation in the Mja KlbA intein is efficient in comparison with those of other intein systems. Interestingly, it also appears that this intermediate is protected against thiolysis by DTT, in contrast to other inteins. Evidence is presented in support of a tight coupling between the N-terminal and C-terminal cleavage steps, despite the fact that the C-terminal single-cleavage reaction occurs in variant Mja KlbA inteins in the absence of N-terminal cleavage. We posit that the splicing events in the Mja KlbA system are tightly coordinated by a network of intra- and interdomain noncovalent interactions, rendering its function particularly sensitive to minor disruptions in the intein or extein environments.

Inteins are the protein equivalent of introns. Their protein splicing activity is essential for the host protein's maturation and function. Inteins are grouped into three classes based on sequence signature and splicing mechanism. The sequence signature of the recently characterized class 3 inteins is a noncontiguous Trp-Cys-Thr (WCT) motif and the absence of the standard class 1 Cys¹ or Ser¹ N-terminal nucleophile. The intein N-terminal Cys¹ or Ser¹ residue is essential for splicing in class 1 inteins. The mycobacteriophage Catera Gp206, Nocardioides sp. strain JS614 TOPRIM, and Thermobifida fusca YX Tfu2914 inteins have a mixture of class 1 and class 3 motifs. They carry the class 3 Trp-Cys-Thr motif and have the standard class 1 N-terminal Ser¹ or Cys¹. This study determined which class the mycobacteriophage Catera Gp206 and Nocardioides sp. JS614 TOPRIM inteins belong to based on catalytic mechanism. The mycobacteriophage Catera Gp206 intein (starting with Ser¹) is a class 3 intein, and its Ser¹ residue is not required for splicing. Based on phylogenetic analysis, we propose that class 3 inteins arose from a single mutated intein that was spread by phage into predominantly helicase genes in various phages and their hosts.

An Arthrobacter species FB24 gene (locus tag Arth_1007) was previously annotated as a putative intein-containing DnaB helicase of phage origin (Arsp-FB24 DnaB intein). However, it is not a helicase gene because the sequence similarity is limited to inteins. In fact, the flanking exteins total only 66 amino acids. Therefore, the intein should be referred to as the Arsp-FB24 Arth_1007 intein. The Arsp-FB24 Arth_1007 intein failed to splice in its native precursor and in a model precursor. We previously noted that the Arsp-FB24 Arth_1007 intein is the only putative Class 3 intein that is missing the catalytically essential Cys at position 4 of intein Motif F, which is one of the three defining signature residues of this class. Additionally, a catalytically essential His in position 10 of intein Motif B is also absent; this His is the most conserved residue amongst all inteins. Splicing activity was not rescued when these two catalytically important positions were 'reverted' back to their consensus residues. This study restores the unity of the Class 3 intein signature sequence in active inteins by demonstrating that the Arsp-FB24 Arth_1007 intein is an inactive pseudogene.

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 Ala(1) 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.

The judicious application of intein technologies to biological problems has resulted in powerful tools for biomedical research. Inteins are intervening sequences that excise themselves from precursor proteins and ligate the surrounding sequences. Variations of intein chemistry have been used to create tagless protein purification strategies, specifically label expressed proteins for biochemical assays, design biosensors, produce microarrays, and synthesize cyclic peptide libraries for inhibitor studies. Moreover, recent advances in small molecule triggered protein splicing allow for tunable post-translational control of protein function in vivo. Inteins are now positioned as an essential tool to study the mechanism of disease progression and validate drug candidates. Yet these tiny proteins have more tricks to play. Recent progress in gene therapy and drug targeting suggest a bright future where split inteins mediate in vivo reconstruction of large therapeutic proteins and target drugs to a specified site of action. Inteins are rapidly becoming valuable tools for drug discovery and drug delivery.

The backbone and side chain resonance assignments of a precursor of the KlbA intein from Methanococcus jannaschii have been determined, based on triple-resonance experiments with the uniformly [13C,15N]-labeled protein.

Certain proteins of unicellular organisms are translated as precursor polypeptides containing inteins (intervening proteins), which are domains capable of performing protein splicing. These domains, in conjunction with a single residue following the intein, catalyze their own excision from the surrounding protein (extein) in a multistep reaction involving the cleavage of two intein-extein peptide bonds and the formation of a new peptide bond that ligates the two exteins to yield the mature protein. We report here the solution NMR structure of a 186-residue precursor of the KlbA intein from Methanococcus jannaschii, comprising the intein together with N- and C-extein segments of 7 and 11 residues, respectively. The intein is shown to adopt a single, well-defined globular domain, representing a HINT (Hedgehog/Intein)-type topology. Fourteen beta-strands are arranged in a complex fold that includes four beta-hairpins and an antiparallel beta-ribbon, and there is one alpha-helix, which is packed against the beta-ribbon, and one turn of 3(10)-helix in the loop between the beta-strands 8 and 9. The two extein segments show increased disorder, and form only minimal nonbonding contacts with the intein domain. Structure-based mutation experiments resulted in a proposal for functional roles of individual residues in the intein catalytic mechanism.

The bacterial phosphoenolpyruvate (PEP):glycose phosphotransferase system (PTS) mediates uptake/phosphorylation of sugars. The transport of all PTS sugars requires Enzyme I (EI) and a phosphocarrier histidine protein of the PTS (HPr). The PTS is stringently regulated, and a potential mechanism is the monomer/dimer transition of EI, because only the dimer accepts the phosphoryl group from PEP. EI monomer consists of two major domains, at the N and C termini (EI-N and EI-C, respectively). EI-N accepts the phosphoryl group from phospho-HPr but not PEP. However, it is phosphorylated by PEP(Mg(2+)) when complemented with EI-C. Here we report that the phosphotransfer rate increases approximately 25-fold when HPr is added to a mixture of EI-N, EI-C, and PEP(Mg(2+)). A model to explain this effect is offered. Sedimentation equilibrium results show that the association constant for dimerization of EI-C monomers is 260-fold greater than the K(a) for native EI. The ligands have no detectable effect on the secondary structure of the dimer (far UV CD) but have profound effects on the tertiary structure as determined by near UV CD spectroscopy, thermal denaturation, sedimentation equilibrium and velocity, and intrinsic fluorescence of the 2 Trp residues. The binding of PEP requires Mg(2+). For example, there is no effect of PEP on the T(m), an increase of 7 degrees C in the presence of Mg(2+), and approximately 14 degrees C when both are present. Interestingly, the dissociation constants for each of the ligands from EI-C are approximately the same as the kinetic (K(m)) constants for the ligands in the complete PTS sugar phosphorylation assays.

Intein-mediated protein splicing is a self-catalytic process in which the intervening intein sequence is removed from a precursor protein and the flanking extein segments are ligated with a native peptide bond. Splice junction proximal residues and internal residues within the intein direct these reactions. The identity of these residues varies in each intein, as groups of related residues populate conserved motifs. Although the basics of the four-step protein splicing pathway are known, mechanistic details are still unknown. Structural and kinetic analyses are beginning to shed some light. Several structures were reported for precursor proteins with mutations in catalytic residues, which stabilize the precursors for crystallographic study. Progress is being made despite limitations inherent in using mutated precursors. However, no uniform mechanism has emerged. Kinetic parameters were determined using conditional trans-splicing (splicing of split precursor fragments after intein reassembly). Several groups concluded that the rate of the initial acyl rearrangement step is rapid and Asn cyclization (step 3) is slow, suggesting that this latter step is rate limiting. Understanding the protein splicing pathway has allowed scientists to harness inteins for numerous applications.

Intein-mediated protein splicing is facilitated by four separate but coordinated nucleophilic displacement reactions that result in the excision of the intein and the ligation of the flanking polypeptides, called the exteins. These reactions are catalyzed by the intein plus the first downstream extein amino acid without the assistance of cofactors or auxiliary enzymes. Non-canonical inteins missing conserved nucleophilic residues at the N- or C-terminus likely splice using variations of the standard mechanism.

Inteins are protein splicing elements that employ standard enzyme strategies to excise themselves from precursor proteins and ligate the surrounding sequences (exteins). The protein splicing pathway consists of four nucleophilic displacements directed by the intein plus the first C-extein residue. The intein active site(s) are formed by folding of the intein within the precursor, which brings together the splice junctions and internal intein residues that assist catalysis. Inteins with non-canonical catalytic residues splice by modified pathways. Understanding intein proteolytic cleavage and ligation activities has led to the development of many novel applications in the fields of protein engineering, enzymology, microarray production, target detection and activation of transgenes in plants. Recent advances include intein-mediated attachment of proteins to solid supports for microarray or western blot analysis, linking nucleic acids to proteins and controllable splicing, which converts inteins into molecular switches., (IUBMB Life, 57: 469-476, 2005.)

A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing. The effectiveness of the system was tested by screening for thermosensitive splicing mutants. Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins.

The self-catalytic protein splicing mechanism is mediated by the intein plus the first amino acid following the intein C-terminus (termed the +1 residue). Although polymorphisms of conserved residues elsewhere in inteins have been widely reported, no splicing-competent intein has been observed without a Ser, Thr or Cys in this functionally essential +1 position. This residue is the nucleophile in two steps of the protein splicing pathway: ligation of the extein fragments during transesterification and formation of a peptide bond between the exteins by an acyl rearrangement. An intein-like element in a hypothetical protein (gene Magn8951) from Magnetospirillum magnetotacticum has all intein signature sequences except the +1 residue, where it has a Tyr. Although the Tyr side-chain hydroxyl can potentially mediate the transesterification reaction, an acyl shift has never been observed with this residue. When the activities of this bacterial intein-like element were studied, protein splicing was not observed and N-terminal cleavage predominated. Mutation of Tyr+1 to Phe or Ala indicated that the Tyr side-chain hydroxyl was not necessary for N-terminal cleavage. Protein splicing activity could be rescued by "reversion" of Tyr+1 to Cys.

Adjacent intein fragments fused to a Snf2/Rad54 helicase-related protein and Snf2/Rad54 helicase were reported for Deinococcus radiodurans R1, leading to the speculation that a frameshift was required for splicing or that trans splicing occurred. However, a type strain (ATCC 13939, RF18410) yielded a single protein that splices by the Ala1 protein splicing pathway, with splicing dependent on adjacent residues.

The majority of inteins are comprised of a protein splicing domain and a homing endonuclease domain. Experimental evidence has demonstrated that the splicing domain and the endonuclease domain in a bifunctional intein are largely independent of each other with respect to both structure and activity. Here, an artificial bifunctional intein has been created through the insertion of an existing homing endonuclease into a mini-intein that is naturally lacking this functionality. The gene for I-CreI, an intron-encoded homing endonuclease, was grafted into the monofunctional Mycobacterium xenopi GyrA intein at the putative site of the missing endonuclease. The resulting fusion protein was found to be capable of protein splicing similar to that of the parent intein. In addition, the protein demonstrated site-specific endonuclease activity that is characteristic of the I-CreI homing endonuclease. The function of each domain therefore remained unaffected by the presence of the other domain. This artificial fusion of the two domains is a potential novel mobile genetic element.

An intein-based positive genetic selection system was developed to study protein splicing and to provide a selection system with the potential for finding splicing inhibitors. Inteins can be novel antimicrobial targets when present in essential proteins since blocking splicing would kill the organism. For example, pathogenic mycobacteria encode inteins that interrupt DNA gyrase. The gyrase selection system exploits (1) splicing of inteins out of Gyrase A and (2) the dominant lethal effect of quinolone poisoning of DNA gyrase, which in turn blocks replication. The system was adapted for whole-cell high-throughput screening using green fluorescent protein as an automatable readout of viability. To demonstrate the efficacy of this system, mutations that blocked splicing of the Mycobacterium xenopi Gyrase A intein were isolated. Splicing was then assayed at a second temperature to identify inteins with a temperature-sensitive splicing phenotype. Mutations were mapped onto a structure-based sequence alignment, which led to the rational prediction of a temperature-sensitive splicing mutation. GyrA intein subdomain relationships also provided insight into intein evolution.

Inteins are self-splicing protein elements (134 to 608 amino acids). Over 125 inteins have been cataloged in InBase, the on-line intein database (http://www.neb.com/neb/inteins.html), which includes the Intein Registry[1]. Inteins naturally present in pathogenic microbes represent novel, yet unexploited drug targets. Understanding the chemistry of the splicing reaction has allowed the manipulation of inteins, which are now used in many protein engineering applications[2].

Plasmodium vivax merozoite surface protein-9 (Pvmsp-9) is characterized here along with orthologues from the related simian malarias Plasmodium cynomolgi and Plasmodium knowlesi. We show that although the corresponding MSP-9 proteins do not have acidic-basic repeated amino acid (aa) motifs, they are related to the Plasmodium falciparum acidic-basic repeat antigen (ABRA) also known as p101. Recognition of this new interspecies Plasmodium MSP family stems from the prior identification of related MSP termed PvMSP-185, PcyMSP-150, and PkMSP-110 on the surface of P. vivax, P. cynomolgi and P. knowlesi merozoites. A clone containing the nearly complete P. knowlesi gene encoding PkMSP-110/MSP-9 provided a hybridization probe and initial sequence information for the design of primers to obtain the P. vivax and P. cynomolgi orthologues using polymerase chain reaction (PCR) amplification strategies. The P. vivax, P. cynomolgi and P. knowlesi msp-9 genes encode proteins that range in calculated molecular mass from 80 to 107 kDa, have typical eukaryotic signal peptides and diverse repeated motifs present immediately upstream of their termination codon. Another feature conserved among these proteins, including the P. falciparum ABRA protein, is the positions of four cysteine residues near the N-terminus, suggesting this conservation maintains structural and perhaps functional characteristics in the MSP-9 family. Rabbit polyclonal antisera raised against recombinantly expressed N-termini of P. knowlesi and P. vivax MSP-9 cross-react with the counterpart proteins in immunofluorescence and immunoblot assays. Comparative interspecies investigations of the potential role(s) of Plasmodium MSP-9 in merozoite invasion of erythrocytes and as a malaria vaccine candidate can now be pursued.

Inteins are self-catalytic protein splicing elements. InBase (http://www.neb.com/neb/inteins.html), the Intein Database and Registry, is a curated compilation of published and unpublished information about protein splicing. It presents general information as well as detailed data for each intein, including tabulated comparisons and a comprehensive bibliography. An intein-specific BLAST server is now available to assist in identifying new inteins.

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.

Protein splicing elements, termed inteins, provide a fertile source for innovative biotechnology tools. First harnessed for protein purification, inteins are now used to express cytotoxic proteins, to segmentally modify or label proteins, to cyclize proteins or peptides, to study structure-activity relationships and to generate reactive polypeptide termini in expressed proteins for an expanding list of chemoselective reactions, including protein ligation.

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.

Protein splicing, the protein equivalent of RNA splicing, is a newly discovered posttranslational process that proceeds through a branched protein intermediate and produces two separate polypeptides from one gene. The experimental data used to distinguish among the proposed protein-splicing mechanisms are presented along with the progress made towards fully describing the mechanism. Numerous protein engineering applications using modified inteins have been developed, including the generation of alpha-thioesters in proteins, which circumvent the limits of solid-phase peptide synthesis.

InBase, the Intein Database (http://www.neb.com/neb/inteins.html ), is a comprehensive on-line resource that includes the Intein Registry. Inteins are protein splicing elements that mediate a self-catalytic protein splicing reaction. InBase presents general information as well as detailed data for each intein, including tabu-lated comparisons and a comprehensive bibliography.

The Mycobacterium xenopi gyrase A mini-intein has been engineered to yield a controllable N-terminal or C-terminal, single-splice-junction autocleavage element. When combined with an affinity tag, these modified mini-inteins can be used to purify target proteins after a single combined chromatography/cleavage step. Cleavage at the intein N terminus was induced with thiol reagents, while cleavage at the intein C terminus was induced by a temperature shift to 16 degrees-25 degrees C. Different preferences for the residue immediately preceding the intein were observed during thiol-induced, N-terminal splice-junction cleavage of the M. xenopi gyrase A mini-intein vs. the Saccharomyces cerevisiae vacuolar ATPase, subunit A (VMA) intein present in the IMPACT purification system. Furthermore, the M. xenopi gyrase A mini-intein C-terminal autocleavage vector allows isolation of polypeptides with N-terminal cysteine residues that are active in the Intein Mediated Protein Ligation method of protein semisynthesis.

Inteins are intervening sequences that splice as proteins, not RNA. InBase, the New England Biolabs Intein Database (http://www.neb. com/neb/inteins.html), is a comprehensive on-line database that includes the Intein Registry, along with detailed information about each intein and its host protein, tabulated comparisons and a comprehensive bibliography including papers in press.

Inteins are protein splicing elements that mediate their excision from precursor proteins and the joining of the flanking protein sequences (exteins). In this study, protein splicing was controlled by splitting precursor proteins within the Psp Pol-1 intein and expressing the resultant fragments in separate hosts. Reconstitution of an active intein was achieved by in vitro assembly of precursor fragments. Both splicing and intein endonuclease activity were restored. Complementary fragments from two of the three fragmentation positions tested were able to splice in vitro. Fragments resulting in redundant overlaps of intein sequences or containing affinity tags at the fragmentation sites were able to splice. Fragment pairs resulting in a gap in the intein sequence failed to splice or cleave. However, similar deletions in unfragmented precursors also failed to splice or cleave. Single splice junction cleavage was not observed with single fragments. In vitro splicing of intein fragments under native conditions was achieved using mini exteins. Trans-splicing allows differential modification of defined regions of a protein prior to extein ligation, generating partially labeled proteins for NMR analysis or enabling the study of the effects of any type of protein modification on a limited region of a protein.

Using two recombinant filarial protein Ags and keyhole limpet hemocyanin, we sensitized T cells from uninfected, nonatopic individuals in such a manner that they were able to provide help for the selective induction of an Ag-specific Ab response. IL-2 and IL-4 were shown to be critical for sensitizing the T cells; once sensitized, these T cells could provide the necessary signals for B cells to produce Ag-specific Abs, provided that IL-4 (or IL-2) was supplied exogenously. Primary exposure of T cells to IFN-gamma, but not to IL-12, prevented the Ag-sensitized T cells from helping B cells to produce specific Abs, apart from the IgG2 isotype. These data suggest that Ab-producing B cells of a defined Ag specificity and isotype can be generated differentially after in vitro priming of human T cells by Ag, providing regulatory cytokines are also present.

The 198-amino-acid in-frame insertion in the gyrA gene of Mycobacterium xenopi is the smallest known naturally occurring active protein splicing element (intein). Comparison with other mycobacterial gyrA inteins suggests that the M. xenopi intein underwent a complex series of events including (i) removal of 222 amino acids that encompass most of the central intein domain, and (ii) addition of a linker of unrelated residues. This naturally occurring genetic rearrangement is a representative characteristic of the taxon. The deletion process removes the conserved motifs involved in homing endonuclease activity. The linker insertion represents a structural requirement, as its mutation resulted in failure to splice. The M. xenopi GyrA intein thus provides a paradigm for a minimal protein splicing element.

It has generally been assumed that the conversion of all inactive protein precursors to biologically active proteins is mediated by specific processing enzymes. However, numerous examples of self-catalyzed protein rearrangements have recently been discovered, including protein splicing and autoproteolysis of hedgehog proteins, glycosylasparaginases and pyruvoyl enzyme precursors. The initial formation of an ester bond by the acyl rearrangement of a peptide bond is a common feature of all of these autoprocessing reactions, which manifest themselves in diverse biological functions, which manifest themselves in diverse biological functions ranging from protein splicing to protein targeting, proenzyme activation, and the generation of enzyme-bound prosthetic groups. Although such acyl rearrangements are thermodynamically unfavorable, their coupling to diverse types of self-catalyzed irreversible steps drives the protein rearrangements to completion.

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

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.

Canlas and coworkers [Canlas et al. (1984) Am. J. Trop. Med. Hyg. 33, 420-424] isolated a monoclonal antibody (MF1) which, upon passive transfer, led to the clearance of Brugia malayi (Bm) microfilariae (mf) from infected jirds. The target of MF1 is a developmentally regulated mf chitinase (Cht) (Fuhrman et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1548-1552). This paper describes the production of enzymatically active Bm Cht in Escherichia coli. Standard expression conditions resulted in production of an insoluble maltose-binding protein (MBP)::Cht fusion protein, but by optimizing expression conditions, the amount of soluble MBP::Cht was increased 25-fold. The specific activity of the soluble MBP::Cht isolated from the E. coli cytoplasm was low. Exporting MBP::Cht into the E. coli periplasmic space increased the specific activity by 12-fold. This suggests that secretion through the membrane and/or the environment of the periplasmic space results in improved folding of recombinant Bm Cht.

Protein splicing results in the expression of two mature proteins from a single gene. After synthesis of a precursor protein, an internal segment (the intein) is excised and the external domains are joined together. A self-catalyzed mechanism for this cleavage-ligation reaction is presented, based on mutagenesis data and analysis of splicing intermediates. Mutations were used to block various steps in the protein splicing pathway, allowing each isolated step to be studied independently. A linear ester intermediate was identified and functional roles for the four conserved splice junction residues were determined. Understanding the mechanism of protein splicing provides a basis for protein engineering studies. For example, inteins can be constructed which fail to splice, but instead cleave the peptide bond at a chosen splice junction.

Protein splicing involves the excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N- and C-terminal regions. It occurs in mesophilic bacteria, yeast, and thermophilic archaea. The ability to control protein splicing of a thermophilic intein by temperature and pH in a foreign protein context facilitated the study of the mechanism of protein splicing in thermophiles. On the other hand, no direct studies have been done on the mechanism of protein splicing in mesophiles. We examined the splicing of a chimeric protein containing the intein of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae that involves cysteines rather than serines at the reaction center. The steps in the splicing process were deduced by analyzing intermediates and side products that accumulated as a result of amino acid substitutions and were found to be analogous to those occurring in thermophiles. Moreover, appropriate amino acid replacements allowed us to develop the first mesophilic in vitro protein splicing system as well as strategies for modulating the rate of protein splicing and for converting the splicing reaction to an efficient protein cleavage reaction at either splice junction.

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.

Dirofilaria immitis is an important filarial parasite of dogs and cats, and a useful model for human filariasis. Current diagnostic tests for heartworm infection in animals rely on the presence of fecund female worms (usually found 6.5 months post-infection or later) and therefore fail to detect pre-patent infections. Putative pepsin inhibitors from 2 filarial parasites of humans namely Onchocerca volvulus (Ov33, Oc3.6, OvD5B) and Brugia malayi (Bm33), have been shown to be useful in diagnosis of onchocerciasis and lymphatic filariasis, respectively. Previous studies have suggested that a homologue exists in D. immitis (DiT33), which may have potential in diagnosis of heartworm infection. In this study, the isolation and characterization of a cDNA clone encoding DiT33 is described. This cDNA contains 12 bases of the nematode-specific 22 nucleotide spliced leader sequence and encodes a 26.4 kDa-protein with a high level of similarity (87-89%) to other filarial members of the family. DiT33 was over-expressed in E. coli as a fusion with the maltose-binding protein and serological analysis was performed using a panel of clinically defined dog sera. The findings of this study indicate that DiT33 is a promising antigen for the early detection of D. immitis and may be a valuable tool in the control and management of heartworm infection.