Hox gene-specific cellular targeting using split intein Trojan exons.

The Trojan exon method, which makes use of intronically inserted T2A-Gal4 cassettes, has been widely used in Drosophila to create thousands of gene-specific Gal4 driver lines. These dual-purpose lines provide genetic access to specific cell types based on their expression of a native gene while simultaneously mutating one allele of the gene to enable loss-of-function analysis in homozygous animals. While this dual use is often an advantage, the truncation mutations produced by Trojan exons are sometimes deleterious in heterozygotes, perhaps by creating translation products with dominant negative effects. Such mutagenic effects can cause developmental lethality as has been observed with genes encoding essential transcription factors. Given the importance of transcription factors in specifying cell type, alternative techniques for generating specific Gal4 lines that target them are required. Here, we introduce a modified Trojan exon method that retains the targeting fidelity and plug-and-play modularity of the original method but mitigates its mutagenic effects by exploiting the self-splicing capabilities of split inteins. "Split Intein Trojan exons" (siTrojans) ensure that the two truncation products generated from the interrupted allele of the native gene are trans-spliced to create a full-length native protein. We demonstrate the efficacy of siTrojans by generating a comprehensive toolkit of Gal4 and Split Gal4 lines for the segmentally expressed Hox transcription factors and illustrate their use in neural circuit mapping by targeting neurons according to their position along the anterior-posterior axis. Both the method and the Hox gene-specific toolkit introduced here should be broadly useful.

Split Gp41-1 intein splicing as a model to evaluate the cellular location of the oncosuppressor Maspin in an in vitro model of osteosarcoma.

Inteins are proteins involved in the protein splicing mechanism, an autoprocessing event, where sequences (exteins) separated by inteins become ligated each other after recombination. Two kinds of inteins have been described, contiguous inteins and split inteins. The former ones are transcribed and translated as a single peptide along with their exteins, while the latter are fragmented between two different genes and are transcribed and translated separately. The aim of this study is to establish a method to obtain a fluorescent eukaryotic protein to analyze its cellular localization, using the natural split gp41-1 inteins. We chose natural split inteins due to their distribution in all three domains of life. Two constructs were prepared, one containing the N-terminal split intein along with the N-moiety of the Red Fluorescent Protein (RFP) and a second construct containing the C-terminal of split intein, the C-moiety of RFP and the gene coding for Maspin, a tumor suppressor protein. The trans-splicing was verified by transfecting both N-terminal and C-terminal constructs into mammalian cells. The success of the recombination event was highlighted through the fluorescence produced by reconstituted RFP after recombination, along with the overlap of the red fluorescence produced by recombined RFP and the green fluorescence produced by the hybridization of the recombinant Maspin with a specific antibody. In conclusion, we opted to use this mechanism of recombination to obtain a fluorescent Maspin instead to express a large fusion protein, considering that it could interfere with Maspin's structure and function.

Site-directed display of zearalenone lactonase on spilt-intein functionalized nanocarrier for green and efficient detoxification of zearalenone.

In this study, we prepared a functional organic-inorganic hybrid nanoflower (InHNF) via split intein moiety in a biomineralization process without using organic solvents. InHNF could specifically bind the target enzymes from crude cell lysates within seconds and site-directedly display them on the surface by forming a peptide bond with enzyme's terminal amino acid residue. This unique feature enabled InHNF to increase the specific activity of zearalenone detoxifying enzyme ZHD518 by 40 âˆ¼ 60% at all tested temperatures and prevented enzyme denaturation even under extreme pH conditions (pH 3-11). Furthermore, it exhibited excellent operational stability, with a residual activity of over 70% after eight reaction cycles. Strikingly, InHNF-ZHD518 achieved above 50% ZEN degradation despite the near inactivation of free ZHD518 in beer sample. Overall, InHNF nanocarriers can achieve environmentally friendly, purification-free, and site-directed immobilization of food enzymes and enhance their catalytic properties, making them suitable for a wide range of industrial applications.

An intein-based biosensor to measure protein stability in vivo.

Biosensors to measure protein stability in vivo are valuable tools for a variety of applications. Previous work has demonstrated that a tripartite design, whereby a protein of interest (POI) is inserted within a reporter, can link POI stability to reporter activity. Inteins are translated within other proteins and excised in a self-mediated protein splicing reaction. Here, we developed a novel folding biosensor where a POI is inserted within an intein, which is subsequently translated within an antibiotic resistance marker. We showed that protein splicing is required for antibiotic resistance and that housing a stable POI within the intein, compared to an unstable variant, results in a 100,000-fold difference in survival. Further, using a fluorescent protein that matures slowly as the POI, we developed a reporter with two simultaneous readouts for protein folding. Finally, we showed that co-expression of GroEL can significantly increase the activity of both reporters, further verifying that protein folding factors can act on the POI in the biosensor. As a whole, our work provides a new twist on the traditional tripartite approach to measuring protein stability in vivo.

Intein-based thermoregulated meganucleases for containment of genetic material.

Limiting the spread of synthetic genetic information outside of the intended use is essential for applications where biocontainment is critical. In particular, biocontainment of engineered probiotics and plasmids that are excreted from the mammalian gastrointestinal tract is needed to prevent escape and acquisition of genetic material that could confer a selective advantage to microbial communities. Here, we built a simple and lightweight biocontainment system that post-translationally activates a site-specific DNA endonuclease to degrade DNA at 18°C and not at higher temperatures. We constructed an orthogonal set of temperature-sensitive meganucleases (TSMs) by inserting the yeast VMA1 L212P temperature-sensitive intein into the coding regions of LAGLIDADG homing endonucleases. We showed that the TSMs eliminated plasmids carrying the cognate TSM target site from laboratory strains of Escherichia coli at the permissive 18°C but not at higher restrictive temperatures. Plasmid elimination is dependent on both TSM endonuclease activity and intein splicing. TSMs eliminated plasmids from E. coli Nissle 1917 after passage through the mouse gut when fecal resuspensions were incubated at 18°C but not at 37°C. Collectively, our data demonstrates the potential of thermoregulated meganucleases as a means of restricting engineered plasmids and probiotics to the mammalian gut.

A Chemical Counterpart to the Resolution Step of Nature's Intein-Mediated Protein Splicing.

In the course of an attempted total chemical synthesis of the ant insulin-like peptide-2 (ILP2) protein molecule, specific cleavage of a backbone peptide bond in a branched ester-linked polypeptide chain with concomitant peptide splicing was observed. The side reaction was investigated in model compounds. Here, we postulate a chemical mechanism for this novel polypeptide backbone cleavage reaction as a chemical counterpart to the resolution step of biochemical intein-mediated protein splicing.

Near-Infrared Optogenetic Module for Conditional Protein Splicing.

Optogenetics has emerged as a powerful tool for spatiotemporal control of biological processes. Near-infrared (NIR) light, with its low phototoxicity and deep tissue penetration, holds particular promise. However, the optogenetic control of polypeptide bond formation has not yet been developed. In this study, we introduce a NIR optogenetic module for conditional protein splicing (CPS) based on the gp41-1 intein. We optimized the module to minimize background signals in the darkness and to maximize the contrast between light and dark conditions. Next, we engineered a NIR CPS gene expression system based on the protein ligation of a transcription factor. We applied the NIR CPS for light-triggered protein cleavage to activate gasdermin D, a pore-forming protein that induces pyroptotic cell death. Our NIR CPS optogenetic module represents a promising tool for controlling molecular processes through covalent protein linkage and cleavage.

Massive intein content in Anaeramoeba reveals aspects of intein mobility in eukaryotes.

Inteins are self-splicing protein elements found in viruses and all three domains of life. How the DNA encoding these selfish elements spreads within and between genomes is poorly understood, particularly in eukaryotes where inteins are scarce. Here, we show that the nuclear genomes of three strains of Anaeramoeba encode between 45 and 103 inteins, in stark contrast to four found in the most intein-rich eukaryotic genome described previously. The Anaeramoeba inteins reside in a wide range of proteins, only some of which correspond to intein-containing proteins in other eukaryotes, prokaryotes, and viruses. Our data also suggest that viruses have contributed to the spread of inteins in Anaeramoeba and the colonization of new alleles. The persistence of Anaeramoeba inteins might be partly explained by intragenomic movement of intein-encoding regions from gene to gene. Our intein dataset greatly expands the spectrum of intein-containing proteins and provides insights into the evolution of inteins in eukaryotes.

A novel protein purification scheme based on salt inducible self-assembling peptides.

BACKGROUND: Protein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release. RESULTS: In this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72-97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%. CONCLUSION: Our results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation.

A Convenient Self-Removing Affinity Tag Method for the Simple Purification of Tagless Recombinant Proteins.

In this work, we describe a novel self-cleaving affinity tag technology based on a highly modified split-intein cleaving element. In this system, which has recently been commercialized by Protein Capture Science, LLC under the name iCapTagTM , the N-terminal segment of an engineered split intein is covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong binding between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. After unwanted impurities are washed from the resin, cleavage of the target protein is initiated by a shift of the buffer pH from 8.5 to 6.2. As a result, the highly purified tagless target protein is released from the column in the elution step. Alternately, the resin beads can be added directly to cell culture broth or lysate, allowing capture, purification and cleavage of the tagless target protein using a column-free format. These methods result in highly pure tagless target protein in a single step, and can thereby accelerate characterization and functional studies. In this work we demonstrate the single step purification of streptokinase, a fibrinolytic agent, and an engineered recombinant human hemoglobin 1.1 (rHb1.1). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression of high-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Basic Protocol 2: Purification of high-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform Alternate Protocol 1: Expression of low-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Alternate Protocol 2: Purification of low-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform.

Construction of IgG-Fab2 bispecific antibody via intein-mediated protein trans-splicing reaction.

A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart's chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG-Fab2-type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG-Fab2 (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG-Fab2 type bsAb will expand the bsAb production method.

Single-Step Non-Chromatographic Purification of Recombinant Mammalian Proteins Using a Split Intein ELP Tag System.

Glycoprotein therapeutics are currently used by large patient populations and generate significant revenue for the biopharmaceutical industry. These therapeutic proteins are currently purified at industrial scale using individualized processes involving multiple chromatographic steps. In the absence of a viable affinity platform method, the required chromatographic steps are difficult to develop and inevitably lead to significant yield losses. Further, during preclinical development, there is a need for reliable platform technologies capable of performing high-throughput screening for biologic candidates. Although affinity tags can provide a solution to some of these challenges, they require specific affinity resins, and the tag itself can interfere with the target protein characteristics. Fusion protein systems consisting of elastin-like polypeptide (ELP) and self-cleaving split inteins such as Npu DnaE can serve as potential non-chromatographic platform technologies for the single-step purification of tagless glycoproteins expressed in mammalian cells. In this chapter, we demonstrate the use of this technology to obtain highly purified anti-ErbB2 ML39 single-chain variable fragment (scFv) expressed from Expi293F suspension cells.

Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses.

Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for diseases requiring large-gene delivery. To overcome this, dual-AAV vector systems that rely on protein trans-splicing were developed, with the split-intein Npu DnaE among the most-used. However, the reconstitution efficiency of Npu DnaE is still insufficient, requiring higher vector doses. In this work, two split-inteins, Cfa and Gp41-1, with reportedly superior trans-splicing were evaluated in comparison with Npu DnaE by transient transfections and dual-AAV in vitro co-transductions. Both Cfa and Gp41-1 split-inteins enabled reconstitution rates that were over two-fold higher than Npu DnaE and 100% of protein reconstitution. The impact of different vector preparation qualities in split-intein performances was also evaluated in co-transduction assays. Higher-quality preparations increased split-inteins' performances by three-fold when compared to low-quality preparations (60-75% vs. 20-30% full particles, respectively). Low-quality vector preparations were observed to limit split-gene reconstitutions by inhibiting co-transduction. We show that combining superior split-inteins with higher-quality vector preparations allowed vector doses to be decreased while maintaining high trans-splicing rates. These results show the potential of more-efficient protein-trans-splicing strategies in dual-AAV vector co-transduction, allowing the extension of its use to the delivery of larger therapeutic genes.

Verifying AXL and putative proteins as SARS-CoV-2 receptors by DnaE intein-based rapid cell-cell fusion assay.

As the understanding of the mechanisms of SARS-CoV-2 infection continues to grow, researchers have come to realize that ACE2 and TMPRSS2 receptors are not the only way for the virus to invade the host, and that there are many molecules that may serve as potential receptors or cofactors. The functionality of these numerous receptors, proposed by different research groups, demands a fast, simple, and accurate validation method. To address this issue, we here established a DnaE intein-based cell-cell fusion system, a key result of our study, which enables rapid simulation of SARS-CoV-2 host cell infection. This system allowed us to validate that proteins such as AXL function as SARS-CoV-2 spike protein receptors and synergize with ACE2 for cell invasion, and that proteins like NRP1 act as cofactors, facilitating ACE2-mediated syncytium formation. Our results also suggest that mutations in the NTD of the SARS-CoV-2 Delta variant spike protein show a preferential selection for Spike-AXL interaction over Spike-LDLRAD3. In summary, our system serves as a crucial tool for the rapid and comprehensive verification of potential receptors, screening of SARS-CoV-2-neutralizing antibodies, or targeted drugs, bearing substantial implications for translational clinical applications.

Metal regulation of Mycobacterium tuberculosis SufB intein splicing at the host-pathogen crossroad.

Intein sequences self-excise from precursor proteins to generate functional proteins in various organisms. Thus, regulation of intein splicing at the host-pathogen interface can determine the fate of infection by controlling generation of essential proteins in microbes. For instance, Mycobacterium tuberculosis (Mtu) SufB intein splicing is crucial for the functionality of SUF complex. This multiprotein system is the sole pathway for [Fe-S] cluster biogenesis in mycobacteria during oxidative stress and Fe starvation. Although metal toxicity and metal starvation are components of host immunity, correlation of metal stress to Mtu SufB intein splicing is missing till date. Current study examines the splicing and N-terminal cleavage reactions of Mtu SufB precursor protein in presence of micronutrient metal ions like Zn+2, Cu+2, and Fe+3/+2. A known intein splicing inhibitor Pt+4 was also tested to support its proposed role as an anti-TB agent. Mtu SufB precursor protein exhibited significant attenuation of splicing and N-terminal cleavage reactions across different concentration ranges for Pt+4, Cu+2, Zn+2, while Fe+3 interaction resulted in precursor accumulation. UV-Vis spectroscopy, inductively coupled plasma-optical emission spectroscopy (ICP-OES), Tryptophan fluorescence assay, and dynamic light scattering (DLS) techniques analyzed metal-protein interaction. Mutagenesis experiments and Ellman's assay identified plausible metal co-ordination sites within Mtu SufB protein. Analyzing the metal effect on Mtu SufB splicing may provide elemental information about the fate of mycobacterial infection, and a probable mechanism to attenuate intracellular survival of Mtu. Current research hints at the host regulatory mechanism on SufB splicing in its native environment and a likely target for developing next-generation anti-TB drugs.

Efficient splicing of the CPE intein derived from directed evolution of the Cryptococcus neoformans PRP8 intein.

Intein-mediated protein splicing has been widely used in protein engineering; however, the splicing efficiency and extein specificity usually limit its further application. Thus, there is a demand for more general inteins that can overcome these limitations. Here, we study the trans-splicing of CPE intein obtained from the directed evolution of Cne PRP8, which shows that its splicing rate is ~29- fold higher than that of the wild-type. When the +1 residue of C-extein is changed to cysteine, CPE also shows high splicing activity. Faster association and higher affinity may contribute to the high splicing rate compared with wild-type intein. These findings have important implications for the future engineering of inteins and provide clues for fundamental studies of protein structure and folding.

split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80.

The split-Gal4 system allows for intersectional genetic labeling of highly specific cell types and tissues in Drosophila. However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is repressible by Gal80. We demonstrate the potent inducibility of "split-intein Gal4" in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell type-specific genetic drivers based on in silico predictions generated by single-cell RNAseq (scRNAseq) datasets, and we describe an algorithm ("Two Against Background" or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible.

Creating Selenocysteine-Specific Reporters Using Inteins.

The selenium moiety in selenocysteine (Sec) imparts enhanced chemical properties to this amino acid and ultimately the protein in which it is inserted. These characteristics are attractive for designing highly active enzymes or extremely stable proteins and studying protein folding or electron transfer, to name a few. There are also 25 human selenoproteins, of which many are essential for our survival. The ability to create or study these selenoproteins is significantly hindered by the inability to easily produce them. Engineering translation has yielded simpler systems to facilitate site-specific insertion of Sec; however, Ser misincorporation remains problematic. Therefore, we have designed two Sec-specific reporters which promote high-throughput screening of Sec translation systems to overcome this barrier. This protocol outlines the workflow to engineer these Sec-specific reporters, with the application to any gene of interest and the ability to transfer this strategy to any organism.

Extein residues regulate the catalytic function of Spl DnaX intein enzyme by restricting the near-attack conformations of the active-site residues.

Intein enzymes catalyze the splicing of their flanking polypeptide chains and have found tremendous biotechnological applications. Their terminal residues form the catalytic core and participate in the splicing reaction. Hence, the neighboring N- and C-terminal extein residues influence the catalytic rate. As these extein residues vary depending on the substrate identity, we tested the influence of 20 amino acids at these sites in the Spl DnaX intein and observed significant variation of spliced product as well as N- and C-terminus cleavage product formation. We investigated the dependence of these reactions on the extein residues by molecular dynamics (MD) simulations on eight extein variants, and found that the conformational sampling of the active-site residues of the intein enzyme differed among these extein variants. We found that the extein variants that sample higher population of near-attack conformers (NACs) of the active-site residues undergo higher product formation in our activity assays. Ground state conformers that closely resemble the transition state are referred to as NACs. Very good correlation was observed between the NAC populations from the MD simulations of eight extein variants and the corresponding product formation from our activity assays. Furthermore, this molecular detail enabled us to elucidate the mechanistic roles of several conserved active-site residues in the splicing reaction. Overall, this study shows that the catalytic power of Spl DnaX intein enzyme, and most likely other inteins, depends on the efficiency of formation of NACs in the ground state, which is further modulated by the extein residues.

Backbone-Installed Split Intein-Assisted Ligation for the Chemical Synthesis of Mirror-Image Proteins.

Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone-Installed Split Intein-Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L-CfaN and L-CfaC ) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (µM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any "ligation scar" on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets.