Development of a new caged intein for multi-input conditional translation of synthetic mRNA.

mRNA medicines can be used to express therapeutic proteins, but the production of such proteins in non-target cells has a risk of adverse effects. To accurately distinguish between therapeutic target and nontarget cells, it is desirable to utilize multiple proteins expressed in each cell as indicators. To achieve such multi-input translational regulation of mRNA medicines, in this study, we engineered Rhodothermus marinus (Rma) DnaB intein to develop "caged Rma DnaB intein" that enables conditional reconstitution of full-length translational regulator protein from split fragments. By combining the caged Rma DnaB intein, the split translational regulator protein, and target protein-binding domains, we succeeded in target protein-dependent translational repression of mRNA in human cells. In addition, the caged Rma intein showed orthogonality to the previously reported Nostoc punctiforme (Npu) DnaE-based caged intein. Finally, by combining these two orthogonal caged inteins, we developed an mRNA-based logic gate that regulates translation based on the expression of multiple intracellular proteins. This study provides important information to develop safer mRNA medicines.

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.

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.

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.

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.

Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics.

DNA polymerases are essential for nucleic acid synthesis, cloning, sequencing and molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive, but retain their structures, such that the intein excises during a heat pulse delivered at 70-80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component 'hot-start' control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from Thermus aquaticus DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5-6 copies of the tested DNA and RNA targets with >95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.

Split selectable marker systems utilizing inteins facilitate gene stacking in plants.

The ability to stack multiple genes in plants is of great importance in the development of crops with desirable traits but can be challenging due to limited selectable marker options. Here we establish split selectable marker systems using protein splicing elements called "inteins" for Agrobacterium-mediated co-transformation in plants. First, we show that such a split selectable marker system can be used effectively in plants to reconstitute a visible marker, RUBY, from two non-functional fragments through tobacco leaf infiltration. Next, to determine the general applicability of our split selectable marker systems, we demonstrate the utility of these systems in the model plants Arabidopsis and poplar by successfully stacking two reporters eYGFPuv and RUBY, using split Kanamycin or Hygromycin resistance markers. In conclusion, this method enables robust plant co-transformation, providing a valuable tool for the simultaneous insertion of multiple genes into both herbaceous and woody plants efficiently.

Use of the redox-dependent intein system for enhancing production of the cyclic green fluorescent protein.

To expand the reported redox-dependent intein system application, in this work, we used the split intein variant with highly trans-splicing efficiency and minimal extein dependence to cyclize the green fluorescent protein variant reporter in vitro. The CPG residues were introduced adjacent to the intein's catalytic cysteine for reversible formation of a disulfide bond to retard the trans-splicing reaction under the oxidative environment. The cyclized reporter protein in Escherichia coli cells was easily prepared by organic extraction and identified by the exopeptidase digestion. The amounts of extracted cyclized protein reporter in BL21 (DE3) cells were higher than those in hyperoxic SHuffle T7 coexpression system for facilitating the disulfide bond formation. The double His6-tagged precursor was purified for in vitro cyclization of the protein for 3 h. Compared with the purified linear counterpart, the cyclic reporter showed about twofold increase in fluorescence intensity, exhibited thermal and hydrolytic stability, and displayed better folding efficiency in BL21 (DE3) cells at the elevated temperature. Taken together, the developed redox-dependent intein system will be used for producing other cyclic disulfide-free proteins. The cyclic reporter is a potential candidate applied in certain thermophilic aerobes.

Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control.

Coordination of multigene expression is one of the key challenges of metabolic engineering for the development of cell factories. Constraints on translation initiation and early ribosome kinetics of mRNA are imposed by features of the 5'UTR in combination with the start of the gene, referred to as the "gene ramp", such as rare codons and mRNA secondary structures. These features strongly influence the translation yield and protein quality by regulating the ribosome distribution on mRNA strands. The utilization of genetic expression sequences, such as promoters and 5'UTRs in combination with different target genes, leads to a wide variety of gene ramp compositions with irregular translation rates, leading to unpredictable levels of protein yield and quality. Here, we present the Standard Intein Gene Expression Ramp (SIGER) system for controlling protein expression. The SIGER system makes use of inteins to decouple the translation initiation features from the gene of a target protein. We generated sequence-specific gene expression sequences for two inteins (DnaB and DnaX) that display defined levels of protein expression. Additionally, we used inteins that possess the ability to release the C-terminal fusion protein in vivo to avoid the impairment of protein functionality by the fused intein. Overall, our results show that SIGER systems are unique tools to mitigate the undesirable effects of gene ramp variation and to control the relative ratios of enzymes involved in molecular pathways. As a proof of concept of the potential of the system, we also used a SIGER system to express two difficult-to-produce proteins, GumM and CBM73.

A cybergenetic framework for engineering intein-mediated integral feedback control systems.

The ability of biological systems to tightly regulate targeted variables, despite external and internal disturbances, is known as Robust Perfect Adaptation (RPA). Achieved frequently through biomolecular integral feedback controllers at the cellular level, RPA has important implications for biotechnology and its various applications. In this study, we identify inteins as a versatile class of genetic components suitable for implementing these controllers and present a systematic approach for their design. We develop a theoretical foundation for screening intein-based RPA-achieving controllers and a simplified approach for modeling them. We then genetically engineer and test intein-based controllers using commonly used transcription factors in mammalian cells and demonstrate their exceptional adaptation properties over a wide dynamic range. The small size, flexibility, and applicability of inteins across life forms allow us to create a diversity of genetic RPA-achieving integral feedback control systems that can be used in various applications, including metabolic engineering and cell-based therapy.

The Evolutionary History of a DNA Methylase Reveals Frequent Horizontal Transfer and Within-Gene Recombination.

Inteins, often referred to as protein introns, are highly mobile genetic elements that invade conserved genes throughout the tree of life. Inteins have been found to invade a wide variety of key genes within actinophages. While in the process of conducting a survey of these inteins in actinophages, we discovered that one protein family of methylases contained a putative intein, and two other unique insertion elements. These methylases are known to occur commonly in phages as orphan methylases (possibly as a form of resistance to restriction-modification systems). We found that the methylase family is not conserved within phage clusters and has a disparate distribution across divergent phage groups. We determined that two of the three insertion elements have a patchy distribution within the methylase protein family. Additionally, we found that the third insertion element is likely a second homing endonuclease, and that all three elements (the intein, the homing endonuclease, and what we refer to as the ShiLan domain) have different insertion sites that are conserved in the methylase gene family. Furthermore, we find strong evidence that both the intein and ShiLan domain are partaking in long-distance horizontal gene transfer events between divergent methylases in disparate phage hosts within the already dispersed methylase distribution. The reticulate evolutionary history of methylases and their insertion elements reveals high rates of gene transfer and within-gene recombination in actinophages.

Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z.

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Increasing the Scalability of Toxin-Intein Orthogonal Combinations.

Inteins are proteins embedded into host proteins from which they are excised in an autocatalytic reaction. Specifically, split inteins are separated into two independent fragments that reconstitute the host protein during the catalytic process. We recently developed a novel strategy for the specific killing of pathogenic and antibiotic resistant bacteria based on toxin-intein combinations. Bacterial type II toxin-antitoxin systems are protein modules in which the toxin can provoke cell death whereas the antitoxin inhibits toxin activity. Although our previous system was based on a split intein (iDnaE) and the CcdB toxin, we demonstrated that iDnaE is able to reconstitute four different toxins. To expand the applicability of our system by widening the repertoire of toxin-intein combinations for complex set-ups, we introduced a second intein, iDnaX, which was artificially split. We demonstrate that iDnaX is able to reconstitute the four toxins, and we manage to reduce its scar size to facilitate their use. In addition, we prove the orthogonality of both inteins (iDnaE and iDnaX) through a toxin reconstitution assay, thus opening the possibility for complex set-ups based on these toxin-intein modules. This could be used to develop specific antimicrobial and other biotechnological applications.

STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity.

Transcription is a dynamic process. To detect the dynamic relationship among protein clusters of RNA polymerase II and coactivators, gene loci, and transcriptional activity, we insert an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site. By optimizing the individual elements, we develop the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNA polymerase II and BRD4 are observed proximal to the transcription start site of Nanog when the gene is transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 tend to be located near the transcription start site, even without transcription activity. Thus, the STREAMING-tag system reveals the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding transcriptional regulation in living cells.

Methods for Recombinant Production and Purification of Peptides as SUMO-Peptide-Intein Fusion Proteins to Protect from Degradation.

Heterologous expression in Escherichia coli is a commonly used method to produce ribosomally synthesized peptides for further study. This generally requires expression of the target protein with an affinity fusion tag, followed by isolation of the fusion protein from a cellular lysate by affinity purification, and finally by removal of the fusion tag and purification of the desired peptide. Sometimes, however, fusion proteins may be degraded during recombinant expression in E. coli. We recently reported an expression system that sandwiches the target peptide between an N-terminal small ubiquitin-like modifier (SUMO) protein and a C-terminal intein. This SUMO-peptide-intein (SPI) fusion protein protects the central peptide from degradation and can lead to improved peptide yield after purification. In this report, we detail the cloning, expression, and isolation procedures for the SPI fusion system, with comments on conditions that can be optimized for different peptides to obtain maximal yield for each construct. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cloning to construct SPI gene Basic Protocol 2: Expression of SPI fusion proteins in E. coli BL21(DE3) Support Protocol: Optimization of expression and induction conditions Basic Protocol 3: Isolation and purification of SPI fusion proteins with a chitin column Alternate Protocol: Isolation and purification of SPI fusion proteins without chitin.

Novel Split Intein-Mediated Enzymatic Channeling Accelerates the Multimeric Bioconversion Pathway of Ginsenoside.

Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.

[Production of antimicrobial peptide DLP4 in Escherichia coli using an ELP-Intein system].

DLP4 (defensin-like peptide 4) is a novel insect defensin, which has strong antibacterial activity against Gram-positive bacteria and is not susceptible to develop drug resistance. In this study, an elastin-like polypeptide (ELP) and an intein fusion system were used for production and purification of DLP4, which combined the characteristics of the phase transition of ELP and the C-cleavage of the intein. A recombinant expression plasmid pET-ELP-I-DLP4 was constructed and transformed into Escherichia coli. Subsequently, DLP4 was purified by simple centrifugation, alternation of pH and temperature. However, the C-cleavage of the intein occurred unexpectedly during the process of expression and purification. To solve this problem, the intein was split into N-intein (I0N) and C-intein (I0C), and fused with ELP or DLP4 to construct two recombinant expression plasmids pET-ELP-I0N and pET-ELP-I0C-DLP4, respectively. These two plasmids were transformed into E. coli separately. The mixture of the two cultures of E. coli strains restored the C-cleavage activity of the intein. This operation yielded DLP4 of about 1.49 mg/L. Antibacterial test confirmed that the purified DLP4 exhibited expected activity. Thus, this approach can be used as an effective way for DLP4 expression and purification in the prokaryotic system.