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.     

Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution

Inteins are internal protein sequences that post-translationally self-excise and splice together the flanking sequences, the so-called exteins. Natural and engineered inteins have been used in many practical applications. However, inteins are often inefficient or inactive when placed in a non-native host protein and may require the presence of several amino acid residues of the native exteins, which will then remain as a potential scar in the spliced protein. Thus, more general inteins that overcome these limitations are highly desirable. Here we report sequential directed evolution as a new approach to produce inteins with such properties. Random mutants of the Ssp (Synechocystis sp. PCC 6803) DnaB mini-intein were inserted into the protein conferring kanamycin resistance at a site where the parent intein was inactive for splicing. The mutants selected for splicing activity were further improved by iterating the procedure for two more cycles at different positions in the same protein. The resulting improved inteins showed high activity in the positions of the first rounds of selection, in multiple new insertion sites, and in different proteins. One of these inteins, the M86 mutant, which accumulated 8 amino acid substitutions, was also biochemically characterized in an artificially split form with a chemically synthesized N-terminal intein fragment consisting of 11 amino acids. When compared with the unevolved split intein, it exhibited an ～60-fold increased rate in the protein trans-splicing reaction and a K(d) value for the interaction of the split intein fragments improved by an order of magnitude. Implications on the intein structure-function, practical application, and evolution are discussed.     

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.     

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.     

Structural and Dynamical Features of Inteins and Implications on Protein Splicing

Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoe-like pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of intein-based technologies.     

Cisplatin inhibits protein splicing, suggesting inteins as therapeutic targets in mycobacteria

Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC(50) of ～2 μM, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ～40 μM, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria.     

Alternative nucleophilic residues in intein catalysis of protein splicing

Protein-splicing inteins are widespread in nature and have found many applications in protein research and engineering. The mechanism of protein splicing typically requires a nucleophilic amino acid residue at both position 1 (first residue of intein) and position +1 (first residue after intein), however it was not clear whether or how the three different nucleophilic residues (Cys, Ser, and Thr) would work differently at these two positions. To use intein in a target protein of interest, one needs to choose an intein insertion site to have a nucleophilic residue at position +1, therefore it is desirable to know what nucleophilic residue(s) are preferred by different inteins. In this study we began with a statistical analysis of known inteins, which showed an unequal distribution of the three nucleophilic residues at positions 1 and +1, and then subjected six different mini-inteins to site-directed mutagenesis to systematically test the functionality of the three nucleophilic residues at the two positions. At position 1, most natural inteins had Cys and none had Thr. When the Cys at position 1 of the six inteins was mutated to Ser and Thr, the splicing activity was abolished in all except one case. At position +1, Cys and Ser were nearly equally abundant in natural inteins, and they were found to be functionally interchangeable in the six inteins of this study. When the two positions were studied as 1/+1 combination, the Cys/Ser combination was abundant in natural inteins, whereas the Ser/Cys combination was conspicuously absent. Similarly, all of the six inteins of this study spliced with the Cys/Ser combination, whereas none spliced with the Ser/Cys combination. These findings have interesting implications on the mechanism of splicing and the selection of intein insertion sites, and they also produced two rare mini-inteins that could splice with Thr at position +1.     

Kinetic and spectroscopic characterization of native and metal-substituted beta-lactamase from Aeromonas hydrophila AE036

Five new inteins were discovered in a survey of 39 mycobacterial strains that was undertaken to clarify the role of RecA inteins in mycobacteria. They are all inserted at the RecA-b site of the recA gene of Mycobacterium chitae, M. fallax, M. gastri, M. shimodei and M. thermoresistibile and belong to the MleRecA allelic family. Sequence analysis showed that although only M. tuberculosis harbours an intein at the RecA-a site the sequence of the RecA-b site is well conserved between species. Furthermore, the presence of inteins does not correlate with specific characteristics of the species such as pathogenicity or growth rate.     

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.     

Enigmatic Distribution,Evolution, and Function of Inteins

Inteins are mobile genetic elements capable of self-splicing post-translationally. They exist in all three domains of life including in viruses and bacteriophage, where they have a sporadic distribution even among very closely related species. In this review, we address this anomalous distribution from the point of view of the evolution of the host species as well as the intrinsic features of the inteins that contribute to their genetic mobility. We also discuss the incidence of inteins in functionally important sites of their host proteins. Finally, we describe instances of conditional protein splicing. These latter observations lead us to the hypothesis that some inteins have adapted to become sensors that play regulatory roles within their host protein, to the advantage of the organism in which they reside.     

Purification of green fluorescent protein using a two-intein system

A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag. The presence of N- and C-terminal chitin-binding domains allowed the affinity purification by a single-affinity chitin column. After the fusion protein was expressed and immobilized on the affinity column, self-cleavage of the inteins was sequentially induced to release the GFPmut3*. The yield was 2.41 mg from 1 l of bacterial culture. Assays revealed that the purity was up to 98% of the total protein. The fluorescence and circular dichroism spectrum of GFPmut3* demonstrated that the purified protein retains the correctly folded structure and function.     

多种S1断裂内含肽的体内交叉反应

断裂内含肽含有两个独立分离的多肽片段(N端内含肽和C端内含肽),它催化蛋白质反式剪接反应,在蛋白质研究与蛋白质工程中已得到诸多实际应用.在蛋白质反式剪接过程中,内含肽的N端内含肽和C端内含肽通过结构互补特异性地非共价组合.然而,Ssp DnaX S1型断裂内含肽的较大C端内含肽片段近来被发现能够与源自其它内含肽的N端内含肽片段交叉反应,表明蛋白质内含子Ssp DnaX具有结构杂交特征.本研究对另外2种S1型内含肽Rma DnaB和Ssp GyrB的较大C端内含肽与不同S1型断裂内含肽的N 端内含肽交叉反应活性进行分析检测.目的是探讨S1型断裂内含肽的结构杂交特征是否具有普遍性.结果发现,Rma DnaB的S1 C端内含肽能够与Ssp GyrB的S1 N端内含肽交叉反应,却不能与Ssp DnaX的S1 N端内含肽交叉反应;与此相似,Ssp GyrB的S1 C端内含肽能够与Rma DnaB的 S1 N端内含肽交叉反应,却不能与Ssp DnaX的S1 N端内含肽交叉反应.此外,某些交叉反应表现出温度依赖性.这些结果对于内含肽的结构 功能关系以及S1型断裂内含肽的应用研究具有重要的意义.     

定向进化提高微小蛋白质内含子Ter DnaE-3 剪接活性的研究

目前,蛋白质内含子在蛋白质工程领域中得到越来越广泛的应用。为提高微小蛋白质内含子Ter DnaE-3(Trichodesmium erythraeum)在异源宿主中的剪接活性,采用易错PCR技术,通过改变反应体系中dNTP、Mg2+、Mn2+的浓度等手段,借助依赖卡那霉素的蛋白质内含子筛选系统进行筛选。Western印迹结果表明:通过定向进化,其中5号突变体的剪接活性从原始的约20%提高至约85%;9号突变体能够避免发生剪接副反应,即N端断裂反应。氨基酸突变位点与剪接活性变化的相关性分析表明:参与α-helix形成的氨基酸的突变极有可能影响蛋白质内含子的断裂反应,参与β-sheet形成的氨基酸的突变则有可能影响蛋白质内含子结构的紧凑性。通过定向进化提高微小蛋白质内含子Ter DnaE-3在异源宿主中的剪接活性,进一步验证依赖卡那霉素抗性的筛选系统的可行性,为扩大蛋白质内含子的应用范围奠定基础。     

Minimization of a eukaryotic mini-intein

Inteins are internal protein splicing elements that can autocatalytically self-excise from their host protein and ligate the protein flanks (exteins) with a peptide bond. Large inteins comprise independent protein splicing and endonuclease domains whereas mini-inteins lack the central endonuclease domain. To identify mini-intein domains that are essential for protein splicing, deletions were introduced at different sites of the 157-aa PRP8 mini-intein of Penicillium chrysogenum. The removal of eight and six amino acids at two different sites resulted in a functional eukaryotic mini-intein of only 143 aa.     

24 Evolutionary dynamics of inteins in bacteria

Inteins are protein sequences that autocatalytically splice themselves out of protein precursors – analogous to introns – and ligate the flanking regions into a functional protein. Inteins are present in all three kingdoms of life, but have a sporadic distribution. They are found predominantly in proteins involved in DNA replication and repair such as helicases. The distribution of inteins suggests an adaptive function. The evolutionary forces which shaped the observed distribution of inteins are generally unknown. Some authors view inteins only as the selfish elements and argue that frequent horizontal transfer is behind inteins sporadic dissemination (Gogarten et al., 2002). On the other hand, the ancient nature of the inteins and the process of gain/loss could lead to the scattered distribution of inteins among species (Pietrokovski, 2001). It is necessary to note that the exclusively selfish nature of inteins is questionable; recent findings support the hypothesis of possible functional roles of inteins in protein regulation (Callahan et al., 2011). Moreover, both hypotheses were built on a limited number of the intein representatives. The amount of genomic data available for bacteria is enormous and in silico analysis for diverse inteins is warranted. We decided to take advantage of these microbial genomic data and performed comprehensive mining for the inteins using a bioinformatic pipeline. Altogether, 1757 species were analysed from 19 major phyla yielding more than 4500 intein-like sequences. The majority of these bacterial inteins were not described previously. Approximately 55% of the inteins were found in polymerases, helicases, or recombinases (Figure 1). Phylogenetic analysis indicated the complex evolutionary dynamics of inteins which includes horizontal transfers, high evolutionary rates coupled with recurrent gains, and losses. The preponderance of inteins in helicases and reductases is being investigated in terms of functional relevance.     

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.     

Protein splicing of the three Pyrococcus abyssi ribonucleotide reductase inteins

An intein is a polypeptide that interrupts the functional domains of a protein, called the exteins. The intein can facilitate its own excision from the exteins, concomitant with the ligation of the exteins, in a process called protein splicing. The alpha subunit of the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi is interrupted by three inteins in separate insertion sites. Each intein can facilitate protein splicing when over-expressed in Escherichia coli, with affinity domains serving as the exteins. The influence of the N-terminal flanking residue on the efficiency of splicing is specific to each intein. Each intein has a different downstream nucleophilic residue, and cannot tolerate substitution to a residue of lesser or equal nucleophilicity. The influence of the conserved penultimate His also differs between the inteins.     

NMR resonance assignment of DnaE intein from Nostoc punctiforme

DnaE intein from Nostoc punctiforme (Npu) is one of naturally occurring split inteins, which has robust protein splicing activity. Highly efficient trans-splicing activity of NpuDnaE intein could widen various biotechnological applications. However, structural basis of the efficient protein splicing activity is poorly understood. As a first step toward better understanding of protein trans-splicing mechanism, we present the backbone and side-chain resonance assignments of a single chain variant NpuDnaE intein as determined by triple resonance experiments with [¹³C,¹⁵N]-labeled protein.     

Protein splicing

Inteins are internal polypeptide sequences that are posttranslationally excised from a protein precursor by a self-catalyzed protein-splicing reaction. Most of inteins consist of N- and C-terminal protein splicing domain and central endonuclease domain. The endonuclease domain can initiate mobility of the intein gene, this process being named intein homing. This review is focused on the recent data about the structure and function of inteins. Main intein-mediated protein-engineering applications, such as protein purification, ligation and cyclization, new forms of biosensors, are presented.     

Four inteins and three group II introns encoded in a bacterial ribonucleotide reductase gene

A bacterial ribonucleotide reductase gene was found to encode four inteins and three group II introns in the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. The 13,650-bp ribonucleotide reductase gene is divided into eight extein- or exon-coding sequences that together encode a 768-amino acid mature ribonucleotide reductase protein, with 83% of the gene sequence encoding introns and inteins. The four inteins are encoded on the second half of the gene, and each has conserved sequence motifs for a protein-splicing domain and an endonuclease domain. These four inteins, together with known inteins, define five intein insertion sites in ribonucleotide reductase homologues. Two of the insertion sites are 10 amino acids apart and next to key catalytic residues of the enzyme. Protein-splicing activities of all four inteins were demonstrated in Escherichia coli. The four inteins coexist with three group II introns encoded on the first half of the same gene, which suggests a breakdown of the presumed barrier against intron insertion in this bacterial conserved protein-coding gene.