Split Intein Research Articles

A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification

Self-cleaving elastin-like protein (ELP) tags provide a very promising tool for recombinant protein purification. With this method, the target protein is purified by simple ELP-mediated precipitation steps, followed by self-cleavage and removal of the ELP tag. Unfortunately, however, inteins usually experience some level of pre-cleavage during protein expression, which can significantly decrease final yields. In this study, we solve this problem by splitting the intein into two ELP-tagged segments. Each segment is incapable of pre-cleavage alone, but the assembled segments release the target protein rapidly when assembled in vitro. The result is the very tight control of the tag cleaving reaction, combined with the simplicity of the ELP purification method. Using this system, we successfully purified four different sizes of target proteins with final yields comparable to or higher than our original contiguous intein-ELP system. Further, we demonstrate a streamlined split intein method, where cells expressing the tagged intein segments are combined prior to cell lysis, allowing the segments to be co-purified in a single reaction mixture.

Simultaneous assembly of two target proteins using split inteins for live cell imaging

Inteins are protein elements that covalently reassemble proteins from two precursor fragments in a process known as protein splicing. They are commonly used to reassemble a single target protein by protein splicing, but a second target protein can potentially reassemble by intein dimerization. Here, we use the naturally occurring split DnaE intein from Nostoc punctiforme (NpuDnaE) to demonstrate the simultaneous assembly of two target proteins in several examples studied with live cell imaging: yellow fluorescent protein (YFP) with monomeric red fluorescent protein (mRFP), dominant positive mutant of RhoA GTPase with YFP and GCaMP2 Ca(2+) indicator with mRFP. These examples showed the versatility of the strategy along with some interesting attributes: first, the two target proteins are in equal stoichiometry; second, the extent of protein splicing can be reported by a fluorescent protein. In particular, the split GCaMP2 with mRFP could find applications in tissue-specific Ca(2+) imaging in transgenic organisms, where mRFP could control for motion-related intensity changes.

Engineering split intein DnaE from Nostoc punctiforme for rapid protein purification

We report the engineering of a DnaE intein able to catalyze rapid C-terminal cleavage in the absence of N-terminal cleavage. A single mutation in DnaE intein from Nostoc punctiforme PCC73102 (NpuDnaE), Asp118Gly, was introduced based on sequence alignment with a previously engineered C-terminal cleaving intein mini-MtuRecA. This mutation was able to both suppress N-terminal cleavage and significantly elevate C-terminal cleavage efficiency. Molecular modeling suggests that in NpuDnaE Asp118 forms a hydrogen bond with the penultimate Asn, preventing its spontaneous cyclization prior to N-terminal cleavage. Mutation of Asp118 to Gly essentially abolishes this restriction leading to subsequent C-terminal cleavage in the absence of N-terminal cleavage. The Gly118 NpuDnaE mutant exhibits rapid thio-dependent C-terminal cleavage kinetics with 80% completion within 3 h at room temperature. We used this newly engineered intein to develop both column-free and chromatography-based protein purification methods utilizing the elastin-like-polypeptide and chitin-binding protein as removable purification tags, respectively. We demonstrate rapid target protein purification to electrophoretic purity at yields up to 84 mg per liter of Escherichia coli culture.

Protein Trans-Splicing of an Atypical Split Intein Showing Structural Flexibility and Cross-Reactivity

Inteins catalyze a protein splicing reaction to excise the intein from a precursor protein and join the flanking sequences (exteins) with a peptide bond. In a split intein, the intein fragments (IN and IC) can reassemble non-covalently to catalyze a trans-splicing reaction that joins the exteins from separate polypeptides. An atypical split intein having a very small IN and a large IC is particularly useful for joining synthetic peptides with recombinant proteins, which can be a generally useful method of introducing site-specific chemical labeling or modifications into proteins. However, a large IC derived from an Ssp DnaX intein was found recently to undergo spontaneous C-cleavage, which raised questions regarding its structure-function and ability to trans-splice. Here, we show that this IC could undergo trans-splicing in the presence of IN, and the trans-splicing activity completely suppressed the C-cleavage activity. We also found that this IC could trans-splice with small IN sequences derived from two other inteins, showing a cross-reactivity of this atypical split intein. Furthermore, we found that this IC could trans-splice even when the IN sequence was embedded in a nearly complete intein sequence, suggesting that the small IN could project out of the central pocket of the intein to become accessible to the IC. Overall, these findings uncovered a new atypical split intein that can be valuable for peptide-protein trans-splicing, and they also revealed an interesting structural flexibility and cross-reactivity at the active site of this intein.

Circular Permutation Prediction Reveals a Viable Backbone Disconnection for Split Proteins: An Approach in Identifying a New Functional Split Intein

Split-protein systems have emerged as a powerful tool for detecting biomolecular interactions and reporting biological reactions. However, reliable methods for identifying viable split sites are still unavailable. In this study, we demonstrated the feasibility that valid circular permutation (CP) sites in proteins have the potential to act as split sites and that CP prediction can be used to search for internal permissive sites for creating new split proteins. Using a protein ligase, intein, as a model, CP predictor facilitated the creation of circular permutants in which backbone opening imposes the least detrimental effects on intein folding. We screened a series of predicted intein CPs and identified stable and native-fold CPs. When the valid CP sites were introduced as split sites, there was a reduction in folding enthalpy caused by the new backbone opening; however, the coincident loss in entropy was sufficient to be compensated, yielding a favorable free energy for self-association. Since split intein is exploited in protein semi-synthesis, we tested the related protein trans-splicing (PTS) activities of the corresponding split inteins. Notably, a novel functional split intein composed of the N-terminal 36 residues combined with the remaining C-terminal fragment was identified. Its PTS activity was shown to be better than current reported two-piece intein with a short N-terminal segment. Thus, the incorporation of in silico CP prediction facilitated the design of split intein as well as circular permutants.

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.

Mutual synergistic protein folding in split intein

Inteins are intervening protein sequences that undergo self-excision from a precursor protein with the concomitant ligation of the flanking polypeptides. Split inteins are expressed in two separated halves, and the recognition and association of two halves are the first crucial step for initiating trans-splicing. In the present study, we carried out the structural and thermodynamic analysis on the interaction of two halves of DnaE split intein from Synechocystis sp. PCC6803. Both isolated halves (IN and IC) are disordered and undergo conformational transition from disorder to order upon association. ITC (isothermal titration calorimetry) reveals that the highly favourable enthalpy change drives the association of the two halves, overcoming the unfavourable entropy change. The high flexibility of two fragments and the marked thermodynamic preference provide a robust association for the formation of the well-folded IN/IC complex, which is the basis for reconstituting the trans-splicing activity of DnaE split intein.

Ultrafast Protein Splicing is Common among Cyanobacterial Split Inteins: Implications for Protein Engineering

We describe the first systematic study of a family of inteins, the split DnaE inteins from cyanobacteria. By measuring in vivo splicing efficiencies and in vitro kinetics, we demonstrate that several inteins can catalyze protein trans-splicing in tens of seconds rather than hours, as is commonly observed for this autoprocessing protein family. Furthermore, we show that when artificially fused, these inteins can be used for rapid generation of protein α-thioesters for expressed protein ligation. This comprehensive survey of split inteins provides indispensable information for the development and improvement of intein-based tools for chemical biology.

In-Cell Fluorescence Activation and Labeling of Proteins Mediated by FRET-Quenched Split Inteins

Methods to visualize, track, and modify proteins in living cells are central for understanding the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have proven to be extremely useful for in vivo studies of protein function, their utility is inherently limited because their spectral and structural characteristics are interdependent. These limitations have spurred the creation of alternative approaches for the chemical labeling of proteins. We report in this work the use of fluorescence resonance emission transfer (FRET)-quenched DnaE split inteins for the site-specific labeling and concomitant fluorescence activation of proteins in living cells. We have successfully employed this approach for the site-specific in-cell labeling of the DNA binding domain (DBD) of the transcription factor YY1 using several human cell lines. Moreover, we have shown that this approach can be also used for modifying proteins to control their cellular localization and potentially alter their biological activity.

A tunable zinc finger-based framework for Boolean logic computation in mammalian cells

The ability to perform molecular-level computation in mammalian cells has the potential to enable a new wave of sophisticated cell-based therapies and diagnostics. To this end, we developed a Boolean logic framework utilizing artificial Cys2–His2 zinc finger transcription factors (ZF-TFs) as computing elements. Artificial ZFs can be designed to specifically bind different DNA sequences and thus comprise a diverse set of components ideal for the construction of scalable networks. We generate ZF-TF activators and repressors and demonstrate a novel, general method to tune ZF-TF response by fusing ZF-TFs to leucine zipper homodimerization domains. We describe 15 transcriptional activators that display 2- to 463-fold induction and 15 transcriptional repressors that show 1.3- to 16-fold repression. Using these ZF-TFs, we compute OR, NOR, AND and NAND logic, employing hybrid promoters and split intein-mediated protein splicing to integrate signals. The split intein strategy is able to fully reconstitute the ZF-TFs, maintaining them as a uniform set of computing elements. Together, these components comprise a robust platform for building mammalian synthetic gene circuits capable of precisely modulating cellular behavior.

The Development of Methods for the Site-Specific Fluorescent Labeling of Spliceosomal Proteins for use in Single-Molecule Studies

Spliceosomes are multi-megadalton ribonucleoprotein (RNP) complexes responsible for catalyzing the removal of noncoding introns from eukaryotic precursor messenger RNA (pre-mRNA) transcripts and ligating the flanking coding exon sequences to produce a mature messenger RNA (mRNA). Spliceosomal assembly and catalysis require a highly dynamic coordination of proteins and RNAs with the purpose of producing mature mRNA that can be properly transcribed by the ribosome. Due to the complexity of the process and a lack of suitable tools, there is still much unknown about the assembly of the spliceosome. Single molecule fluorescence microscopy tools have recently been developed for studies of spliceosome assembly and dynamics of the pre-mRNA substrate throughout splicing. In an effort to advance the study of splicing and delineate the exact timing and dynamics required to achieve splicing, we are currently investigating methods for the site-specific labeling of proteins, one of these methods being protein trans-splicing (PTS) using a split intein. Protein splicing is a naturally occurring process in which a protein editor, called an intein, excises itself out of a host protein in which it is embedded creating a new peptide bond between its two flanking regions, the exteins. PTS uses an artificially or naturally split intein to create a new peptide bond between flanking exteins similar to protein splicing. Upon incubation of the two components, protein splicing will take place, resulting in the ligation of the two extein regions. By fluorescently labeling one of the extein components, we hope to use PTS to site-specifically label several spliceosomal proteins with the ultimate goal of correlating the association and dissociation of spliceosomal components with pre-mRNA dynamics in order to gain a better understanding of the mechanism of pre-mRNA splicing.

Split-intein mediated re-assembly of genetically encoded Ca2+ indicators

While genetically encoded Ca2+ indicators (GECIs) allow Ca2+ imaging in model organisms, the gene expression is often under the control of a single promoter that may drive expression beyond, the cell types of interest. To enable more cell-type specific targeting, GECIs can be brought under the, control of the intersecting expression from two promoters. Here, we present the splitting and, reassembly of two representative GECIs (TN-XL and GCaMP2) mediated by the split intein from Nostoc, punctiforme (NpuDnaE). While the split TN-XL biosensor offered ratiometric Ca2+ imaging, it had a, diminished Ca2+ response relative to the native TN-XL biosensor. In contrast, the split GCaMP2, biosensor retained similar Ca2+ response to the native GCaMP2. The split GCaMP2 biosensor was, further targeted to the pharyngeal muscles of Caenorhabditis elegans where Ca2+ signals from feeding C. elegans, were imaged. Thus, we envision that increased cell-type targetability of GECIs is feasible with two, complementary promoters.

Split Inteins: Nature's Protein Ligases.

Split inteins carry out a naturally occurring process known as protein trans-splicing, where two protein fragments bind to form a catalytically competent enzyme, then catalyze their own excision and the ligation of their flanking sequences. In the past thirteen years since their discovery, chemists and biologists have utilized split inteins in exogenous contexts for a number of biotechnological applications centered around the formation of native peptide bonds. While many protein trans-splicing technologies have emerged and flourished in recent years, several factors still limit their wide-spread practical use. Here, we discuss the development, applications, and limitations of split intein-based technologies and propose that further advancement in this field will require a more fundamental understanding of split intein structure and function.

Split-Inteins for Simultaneous, site-specific conjugation of Quantum Dots to multiple protein targets In vivo

BackgroundProteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or in vivo. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs) to the C-terminus of target proteins in vivo. In this study, we expand this approach to achieve site-specific conjugation of QDs to two or more proteins simultaneously with spectrally distinguishable QDs for multiparameter imaging of cellular functions.ResultsUsing the DnaE split intein we target QDs to the C-terminus of paxillin and show that paxillin-QD conjugates become localized at focal adhesions allowing imaging of the formation and dissolution of these complexes. We go on to utilize a different split intein, namely Ssp DnaB mini-intein, to demonstrate N-terminal protein tagging with QDs. Combination of these two intein systems allowed us to simultaneously target two distinct proteins with spectrally distinguishable QDs, in vivo, without any cross talk between the two intein systems.ConclusionsMultiple target labeling is a unique feature of the intein based methodology which sets it apart from existing tagging methodologies in that, given the large number of characterized split inteins, the number of individual targets that can be simultaneously tagged is only limited by the number of QDs that can be spectrally distinguished within the cell. Therefore, the intein-mediated approach for simultaneous, in vivo, site-specific (N- and C-terminus) conjugation of Quantum Dots to multiple protein targets opens up new possibilities for bioimaging applications and offers an effective system to target QDs and other nanostructures to intracellular compartments as well as specific molecular complexes.

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.

Spontaneous C-cleavage of a mini-intein without its conserved N-terminal motif A

Previously, the C-terminal fragment of a split intein was known to undergo controllable C-cleavage at its C-terminus only when the N-terminal fragment of the intein was added. Here we constructed a similar split intein from the Ssp DnaX intein, but we unexpectedly observed that its C-terminal 136-aa fragment could undergo spontaneous C-cleavage without the N-terminal fragment that was up to 15 aa long and contained the conserved intein motif A. This C-cleavage activity was significantly decreased by a mutation of the conserved Thr residue in the conserved intein motif B. These findings suggest a robust intein structure in the absence of motif A and a larger role of motif B in the third step of the protein splicing mechanism.

Evolution of cyclic peptide protease inhibitors

We report a bacterial system for the evolution of cyclic peptides that makes use of an expanded set of amino acid building blocks. Orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pairs, together with a split intein system were used to biosynthesize a library of ribosomal peptides containing amino acids with unique structures and reactivities. This peptide library was subsequently used to evolve an inhibitor of HIV protease using a selection based on cellular viability. Two of three cyclic peptides isolated after two rounds of selection contained the keto amino acid p-benzoylphenylalanine (pBzF). The most potent peptide (G12: GIXVSL; X=pBzF) inhibited HIV protease through the formation of a covalent Schiff base adduct of the pBzF residue with the ε-amino group of Lys 14 on the protease. This result suggests that an expanded genetic code can confer an evolutionary advantage in response to selective pressure. Moreover, the combination of natural evolutionary processes with chemically biased building blocks provides another strategy for the generation of biologically active peptides using microbial systems.

Kinetic Control of One-Pot Trans-Splicing Reactions by Using a Wild-Type and Designed Split Intein

Charge-swapping of key intermolecular ion clusters in the naturally split DnaE intein from Nostoc punctiforme (NpuWT) alters split intein binding affinities and trans-splicing kinetics. This concept was used to engineer the new split intein NpuMUT with low cross-reactivity to NpuWT. This intein pair can be used to catalyze multiple trans-splicing reactions in one pot with kinetically controlled selectivity (see picture).

Kinetic Control of One‐Pot Trans‐Splicing Reactions by Using a Wild‐Type and Designed Split Intein

Kinetic Control of One‐Pot Trans‐Splicing Reactions by Using a Wild‐Type and Designed Split Intein

Protein trans-splicing as a protein ligation tool to study protein structure and function.

Protein trans-splicing (PTS) exerted by split inteins is a protein ligation reaction which enables overcoming the barriers of conventional heterologous protein production. We provide an overview of the current state-of-the-art in split intein engineering, as well as the achievements of PTS technology in the realm of protein structure-function analyses, including incorporation of natural and artificial protein modifications, controllable protein reconstitution, segmental isotope labeling and protein cyclization. We further discuss factors crucial for the successful implementation of PTS in these protein engineering approaches, and speculate on necessary future endeavours to make PTS a universally applicable protein ligation tool.