Intein splicing efficiency and RadA levels can control the mode of archaeal DNA replication.

Inteins (intervening proteins), mobile genetic elements removed through protein splicing, often interrupt proteins required for DNA replication, recombination, and repair. An abundance of in vitro evidence implies that inteins may act as regulatory elements, whereby reduced splicing inhibits production of the mature protein lacking the intein, but in vivo evidence of regulatory intein excision in the native host is absent. The model archaeon Thermococcus kodakarensis encodes 15 inteins, and we establish the impacts of intein splicing inhibition on host physiology and replication in vivo. We report that a decrease in intein splicing efficiency of the recombinase RadA, a Rad51/RecA homolog, has widespread physiological consequences, including a general growth defect, increased sensitivity to DNA damage, and a switch in the mode of DNA replication from recombination-dependent replication toward origin-dependent replication.

Improved protein splicing through viral passaging.

Intervening proteins (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flanking sequences (exteins) are joined with a peptide bond. Previously, we developed a self-removing translation reporter for labeling Ebola virus (EBOV). In this reporter, an intein (RadA) containing the fluorescent protein ZsGreen (ZsG) is inserted within the EBOV protein VP30. Upon VP30-RadA-ZsG expression from the viral genome, RadA-ZsG is removed from VP30 through the protein splicing activity of RadA, generating functional, non-tagged VP30 and functional ZsGreen. While incorporation of our VP30-RadA-ZsG fusion reporter into recombinant EBOV (rEBOV-RadA-ZsG) resulted in an infectious virus that expresses ZsG upon infection of cells, this virus displayed a replication defect compared to wild-type EBOV, which might be the result of insufficient RadA splicing. Here, we demonstrate that the serial passaging of rEBOV-RadA-ZsG in human cells led to an increase in replication efficiency compared to unpassaged rEBOV-RadA-ZsG. Sequencing of passaged viruses revealed intein-specific mutations. These mutations improve intein activity in both prokaryotic and eukaryotic systems, as well as in multiple extein contexts. Taken together, our findings offer a novel means to select for inteins with enhanced catalytic properties that appear independent of extein context and expression system.IMPORTANCEIntervening proteins (inteins) are self-removing protein elements that have been utilized to develop a variety of innovative protein engineering technologies. Here, we report the isolation of inteins with improved catalytic activity through viral passaging. Specifically, we inserted a highly active intein within an essential protein of Ebola virus and serially passaged this recombinant virus, which led to intein-specific hyper-activity mutations. The identified mutations showed improved intein activity within both bacterial and eukaryotic expression systems and in multiple extein contexts. These results present a new strategy for developing inteins with improved splicing activity.

Understanding operon architecture using LEGO bricks.

Many undergraduates struggle to interpret abstract concepts in molecular biology. Modeling can facilitate learning by making these abstract concepts tangible. Here, we present an exercise based on the lac operon designed for undergraduate students using LEGO bricks. The lac operon is a classic example of transcriptional regulation taught in a variety of undergraduate biology courses and is fundamental to understanding the regulation of gene expression. This easy-to-implement active learning exercise demonstrates how the various components of the lac operon are oriented under a variety of nutritional conditions to control gene expression. In addition, higher-order concepts, such as the effect of mutation on lac operon expression, can be readily modeled. Overall, students not only found this exercise to be enjoyable but also helpful as a tool to engage with this course material.

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.

Inteins-mechanism of protein splicing, emerging regulatory roles, and applications in protein engineering.

Protein splicing is a posttranslational process in which an intein segment excises itself from two flanking peptides, referred to as exteins. In the native context, protein splicing results in two separate protein products coupled to the activation of the intein-containing host protein. Inteins are generally described as either full-length inteins, mini-inteins or split inteins, which are differentiated by their genetic structure and features. Inteins can also be divided into three classes based on their splicing mechanisms, which differ in the location of conserved residues that mediate the splicing pathway. Although inteins were once thought to be selfish genetic elements, recent evidence suggests that inteins may confer a genetic advantage to their host cells through posttranslational regulation of their host proteins. Finally, the ability of modified inteins to splice and cleave their fused exteins has enabled many new applications in protein science and synthetic biology. In this review, we briefly cover the mechanisms of protein splicing, evidence for some inteins as environmental sensors, and intein-based applications in protein engineering.

An artificially split class 3 intein.

Inteins excise themselves from precursor polypeptides through protein splicing, joining N- and C-exteins with a peptide bond. Split inteins are expressed as separate polypeptides that undergo protein trans splicing (PTS). Here, we demonstrate PTS can be achieved using an artificially split class 3 intein. Because class 3 inteins use an internal initiating nucleophile near the C-extein junction, rather than the first residue of the intein, both catalytic nucleophiles are present on a single polypeptide. This results in a compact arrangement of catalytic nucleophiles for PTS compared to the standard arrangement for split class 1 inteins.

High-yield vesicle-packaged recombinant protein production from E. coli.

We describe an innovative system that exports diverse recombinant proteins in membrane-bound vesicles from E. coli. These recombinant vesicles compartmentalize proteins within a micro-environment that enables production of otherwise challenging insoluble, toxic, or disulfide-bond containing proteins from bacteria. The release of vesicle-packaged proteins supports isolation from the culture and allows long-term storage of active protein. This technology results in high yields of vesicle-packaged, functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine.

Labeling Ebola Virus with a Self-Splicing Fluorescent Reporter.

Inteins (intervening proteins) are polypeptides that interrupt the sequence of other proteins and remove themselves through protein splicing. In this intein-catalyzed reaction, the two peptide bonds surrounding the intein are rearranged to release the intein from the flanking protein sequences, termed N- and C-exteins, which are concurrently joined by a peptide bond. Because of this unique functionality, inteins have proven exceptionally useful in protein engineering. Previous work has demonstrated that heterologous proteins can be inserted within an intein, with both the intein and inserted protein retaining function, allowing for intein-containing genes to coexpress additional coding sequences. Here, we show that a fluorescent protein (ZsGreen) can be inserted within the Pyrococcus horikoshii RadA intein, with the hybrid protein (ZsG-Int) maintaining fluorescence and splicing capability. We used this system to create a recombinant Ebola virus expressing a fluorescent protein. We first tested multiple potential insertion sites for ZsG-Int within individual Ebola virus proteins, identifying a site within the VP30 gene that facilitated efficient intein splicing in mammalian cells while also preserving VP30 function. Next, we successfully rescued a virus containing the ZsG-Int-VP30 fusion protein, which displayed fluorescence in the infected cells. We thus report a new intein-based application for adding reporters to systems without the need to add additional genes. Further, this work highlights a novel reporter design, whereby the reporter is only made if the protein of interest is translated and does not remain fused to the protein of interest.

Intein Inhibitors as Novel Antimicrobials: Protein Splicing in Human Pathogens, Screening Methods, and Off-Target Considerations.

Protein splicing is a post-translational process by which an intervening polypeptide, or intein, catalyzes its own removal from the flanking polypeptides, or exteins, concomitant with extein ligation. Although inteins are highly abundant in the microbial world, including within several human pathogens, they are absent in the genomes of metazoans. As protein splicing is required to permit function of essential proteins within pathogens, inteins represent attractive antimicrobial targets. Here we review key proteins interrupted by inteins in pathogenic mycobacteria and fungi, exciting discoveries that provide proof of concept that intein activity can be inhibited and that this inhibition has an effect on the host organism's fitness, and bioanalytical methods that have been used to screen for intein activity. We also consider potential off-target inhibition of hedgehog signaling, given the similarity in structure and function of inteins and hedgehog autoprocessing domains.

Monitoring Protein Splicing Using In-gel Fluorescence Immediately Following SDS-PAGE.

Inteins garner significant interest from both basic and applied researchers due to their unique catalytic abilities. Herein, we describe a protocol for accurately monitoring protein splicing without purification using in-gel fluorescence immediately following Tris-Glycine SDS-PAGE. Following expression in Escherichia coli, cells are lysed by sonication, cell supernatants are separated using Tris-Glycine SDS-PAGE, and superfolder GFP (sfGFP) fluorescence is directly visualized within gels. This method is rapid, with sfGFP immediately imaged following SDS-PAGE without staining. Further, sfGFP can be specifically detected in complex samples such as E. coli cell supernatants, proteins run at expected masses, and all steps can be performed at ambient temperature. This strategy is broadly applicable beyond the study of protein splicing and can be used for sensitive and specific visualization of superfolder sfGFP-tagged proteins in-gel.

Reactive Chlorine Species Reversibly Inhibit DnaB Protein Splicing in Mycobacteria.

Intervening proteins, or inteins, are mobile genetic elements that are translated within host polypeptides and removed at the protein level by splicing. In protein splicing, a self-mediated reaction removes the intein, leaving a peptide bond in place. While protein splicing can proceed in the absence of external cofactors, several examples of conditional protein splicing (CPS) have emerged. In CPS, the rate and accuracy of splicing are highly dependent on environmental conditions. Because the activity of the intein-containing host protein is compromised prior to splicing and inteins are highly abundant in the microbial world, CPS represents an emerging form of posttranslational regulation that is potentially widespread in microbes. Reactive chlorine species (RCS) are highly potent oxidants encountered by bacteria in a variety of natural environments, including within cells of the mammalian innate immune system. Here, we demonstrate that two naturally occurring RCS, namely, hypochlorous acid (the active compound in bleach) and N-chlorotaurine, can reversibly block splicing of DnaB inteins from Mycobacterium leprae and Mycobacterium smegmatis in vitro. Further, using a reporter that monitors DnaB intein activity within M. smegmatis, we show that DnaB protein splicing is inhibited by RCS in the native host. DnaB, an essential replicative helicase, is the most common intein-housing protein in bacteria. These results add to the growing list of environmental conditions that are relevant to the survival of the intein-containing host and influence protein splicing, as well as suggesting a novel mycobacterial response to RCS. We propose a model in which DnaB splicing, and therefore replication, is paused when these mycobacteria encounter RCS. IMPORTANCE Inteins are both widespread and abundant in microbes, including within several bacterial and fungal pathogens. Inteins are domains translated within host proteins and removed at the protein level by splicing. Traditionally considered molecular parasites, some inteins have emerged in recent years as adaptive posttranslational regulatory elements. Several studies have demonstrated CPS, in which the rate and accuracy of protein splicing, and thus host protein functions, are responsive to environmental conditions relevant to the intein-containing organism. In this work, we demonstrate that two naturally occurring RCS, including the active compound in household bleach, reversibly inhibit protein splicing of Mycobacterium leprae and Mycobacterium smegmatis DnaB inteins. In addition to describing a new physiologically relevant condition that can temporarily inhibit protein splicing, this study suggests a novel stress response in Mycobacterium, a bacterial genus of tremendous importance to humans.

Construction and Quantitation of a Selectable Protein Splicing Sensor Using Gibson Assembly and Spot Titers.

Inteins (intervening proteins) are translated within host proteins and removed through protein splicing. Conditional protein splicing (CPS), where the rate and accuracy of splicing are highly dependent on environmental cues, has emerged as a novel form of post-translational regulation. While CPS has been demonstrated for several inteins in vitro, a comprehensive understanding of inteins requires tools to quantitatively monitor their activity within the cellular context. Here, we describe a method for construction of a splicing-dependent system that can be used to quantitatively assay for conditions that modulate protein splicing. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Construction of an intein-containing KanR2 library using Gibson assembly Basic Protocol 2: Phenotype determination using quantitative spot titers Support Protocol 1: Preparation of LB agar plates for spot titers Support Protocol 2: Preparation and transformation of competent M. smegmatis cells.

Microbioreactor for lower cost and faster optimisation of protein production.

Optimisation of bioprocesses relies on approaches that are either labour intensive or require expensive robotic systems. There is a need for fluidic processing at low volume that can be integrated with existing bioprocess analytics to provide analytical information for the development and optimisation of bioprocesses. We demonstrate a 1 mL polymer inkjet 3D printed (i3DP) microbioreactor with integrated sensing (pH, oxygen and cell density) for optimisation of recombinant protein production with different feeds. A pressurised fluid driving system was used to control flow rates down to 0.7 µL min-1 with fluid switching from four reservoirs using a manifold controlled by solenoid valves. Oxygen transferred from a headspace via a gas-permeable membrane achieved a kLa of up to 90 h-1 at 1500 rpm. Cultivation of E. coli within the microbioreactor was comparable with a 2 L bench scale bioreactor, with optical densities of respectively 7.1 ± 0.4 and 6.5 ± 0.35. Triplicate batch cultivations within the microbioreactor of Pichia pastoris, with diauxic growth on glycerol (0.20 ± 0.02 h-1) and methanol (0.02 ± 0.04 h-1), showed good control of pH and DO and achieved a maximum dry cell weight of 10 ± 1 g L-1. For continuous cultivations, recombinant protein production was higher in pure methanol (314 ± 23) than methanol-sorbitol (202 ± 17) but reduces over time with lower cellular viability for methanol-glucose mixed feed, with less total protein produced and increases in DNA and proteases released. The developed system could be used in different applications including within synthetic biology, cell and gene therapy and organ-on-chips.

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria.

Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc.IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential.

Mechanism of Single-Stranded DNA Activation of Recombinase Intein Splicing.

Inteins, or intervening proteins, are mobile genetic elements translated within host polypeptides and removed through protein splicing. This self-catalyzed process breaks two peptide bonds and rejoins the flanking sequences, called N- and C-exteins, with the intein scarlessly escaping the host protein. As these elements have traditionally been viewed as purely selfish genetic elements, recent work has demonstrated that the conditional protein splicing (CPS) of several naturally occurring inteins can be regulated by a variety of environmental cues relevant to the survival of the host organism or crucial to the invading protein function. The RadA recombinase from the archaeon Pyrococcus horikoshii represents an intriguing example of CPS, whereby protein splicing is inhibited by interactions between the intein and host protein C-extein. Single-stranded DNA (ssDNA), a natural substrate of RadA as well as signal that recombinase activity is needed by the cell, dramatically improves the splicing rate and accuracy. Here, we investigate the mechanism by which ssDNA exhibits this influence and find that ssDNA strongly promotes a specific step of the splicing reaction, cyclization of the terminal asparagine of the intein. Interestingly, inhibitory interactions between the host protein and intein that block splicing localize to this asparagine, suggesting that ssDNA binding alleviates this inhibition to promote splicing. We also find that ssDNA directly influences the position of catalytic nucleophiles required for protein splicing, implying that ssDNA promotes assembly of the intein active site. This work advances our understanding of how ssDNA accelerates RadA splicing, providing important insights into this intriguing example of CPS.

Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein.

BACKGROUND: Many valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the 'hijack' of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant. RESULTS: We report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5'UTR and first 47 amino acidsof FliC from E. coli (but no 3'UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH2)], achieving secreted yields of up to 0.29 mg L-1, in low cell density culture. CONCLUSIONS: We have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.

Mycobacterial DnaB helicase intein as oxidative stress sensor.

Inteins are widespread self-splicing protein elements emerging as potential post-translational environmental sensors. Here, we describe two inteins within one protein, the Mycobacterium smegmatis replicative helicase DnaB. These inteins, DnaBi1 and DnaBi2, have homology to inteins in pathogens, splice with vastly varied rates, and are differentially responsive to environmental stressors. Whereas DnaBi1 splicing is reversibly inhibited by oxidative and nitrosative insults, DnaBi2 is not. Using a reporter that measures splicing in a native intein-containing organism and western blotting, we show that H2O2 inhibits DnaBi1 splicing in M. smegmatis. Intriguingly, upon oxidation, the catalytic cysteine of DnaBi1 forms an intramolecular disulfide bond. We report a crystal structure of the class 3 DnaBi1 intein at 1.95 Å, supporting our findings and providing insight into this splicing mechanism. We propose that this cysteine toggle allows DnaBi1 to sense stress, pausing replication to maintain genome integrity, and then allowing splicing immediately when permissive conditions return.

Conditional Protein Splicing Switch in Hyperthermophiles through an Intein-Extein Partnership.

Inteins are intervening proteins that undergo an autocatalytic splicing reaction that ligates flanking host protein sequences termed exteins. Some intein-containing proteins have evolved to couple splicing to environmental signals; this represents a new form of posttranslational regulation. Of particular interest is RadA from the archaeon Pyrococcus horikoshii, for which long-range intein-extein interactions block splicing, requiring temperature and single-stranded DNA (ssDNA) substrate to splice rapidly and accurately. Here, we report that splicing of the intein-containing RadA from another archaeon, Thermococcus sibericus, is activated by significantly lower temperatures than is P. horikoshii RadA, consistent with differences in their growth environments. Investigation into variations between T. sibericus and P. horikoshii RadA inteins led to the discovery that a nonconserved region (NCR) of the intein, a flexible loop where a homing endonuclease previously resided, is critical to splicing. Deletion of the NCR leads to a substantial loss in the rate and accuracy of P. horikoshii RadA splicing only within native exteins. The influence of the NCR deletion can be largely overcome by ssDNA, demonstrating that the splicing-competent conformation can be achieved. We present a model whereby the NCR is a flexible hinge which acts as a switch by controlling distant intein-extein interactions that inhibit active site assembly. These results speak to the repurposing of the vestigial endonuclease loop to control an intein-extein partnership, which ultimately allows exquisite adaptation of protein splicing upon changes in the environment.IMPORTANCE Inteins are mobile genetic elements that interrupt coding sequences (exteins) and are removed by protein splicing. They are abundant elements in microbes, and recent work has demonstrated that protein splicing can be controlled by environmental cues, including the substrate of the intein-containing protein. Here, we describe an intein-extein collaboration that controls temperature-induced splicing of RadA from two archaea and how variation in this intein-extein partnership results in fine-tuning of splicing to closely match the environment. Specifically, we found that a small sequence difference between the two inteins, a flexible loop that likely once housed a homing endonuclease used for intein mobility, acts as a switch to control intein-extein interactions that block splicing. Our results argue strongly that some inteins have evolved away from a purely parasitic lifestyle to control the activity of host proteins, representing a new form of posttranslational regulation that is potentially widespread in the microbial world.

Inteins.

Lennon and Belfort introduce inteins - protein introns - and describe how they escape host proteins, their uses in biotechnology, where they are found in nature, and their role in post-translational regulation.