Protein Fusion Research Articles

Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates.

Protein modification with non-canonical amino acids (ncAAs) represents a useful technology to afford homogenous samples of bioconjugates with site-specific modification. This technique can be directly applied to the detection of aberrant SUMOylation patterns, which are often indicative of disease states. Modified SUMO-trapping proteins, consisting of a catalytically inactive ULP1 fragment (UTAG) fused to the maltose-binding protein MBP, are useful reagents for the binding and labeling of SUMOylated proteins. Mutation of this UTAG fusion protein to facilitate amber suppression technologies for the genetic incorporation of ncAAs was assessed to provide a functional handle for modification. Ultimately, two sites in the maltose-binding protein (MBP) fusion were identified as ideal for incorporation and bioconjugation without perturbation to the SUMO-trapping ability of the UTAG protein. This functionality was then employed to label SUMOylated proteins in HeLa cells and demonstrate their enrichment in the nucleus. This modified UTAG-MBP-ncAA protein has far-reaching applications for both diagnostics and therapeutics.

Antimicrobial activity of the recombinant peptide Melittin-Thanatin with three glycine to tryptophan mutations

The antimicrobial peptide was considered an important target for developing novel antibacterial drugs. However, the unstable biological activity and the low antibacterial activity are challenges for the application of recombinant proteins. In this study, the fusion peptide of Melittin-Thanatin (MT) was designed and produced, and its derivative sequence (MT-W) was obtained by replacing three glycines (Gly, G) with tryptophan (Trp, W). The MT-W peptide were synthesized in Bacillus subtilis WB700 by EDDIE self-cleavage protein fusion. Compared with MT, MT-W exhibited 2–4 times higher antibacterial rate against Escherichia coli K88. In addition, MT-W showed lower cytotoxicity (IC50 > 300 mg·L−1) to the red blood cell, and more stable biological activities under the conditions of different temperatures (20, 30, 40, 50, 60, 70, 80, and 90 °C), pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) and different proteases. Especially, MT-W showed a broader antibacterial effect on three drug-resistant strains than florfenicol and oxytetracycline calcium. In conclusion, compared with MT, the MT-W showed increased antibacterial activity, stability, lower cytotoxicity, and broader antimicrobial effect. Therefore, it would become a promising alternative to conventional antibiotics.

Dependence of diffusion in Escherichia coli cytoplasm on protein size, environmental conditions, and cell growth.

Inside prokaryotic cells, passive translational diffusion typically limits the rates with which cytoplasmic proteins can reach their locations. Diffusion is thus fundamental to most cellular processes, but the understanding of protein mobility in the highly crowded and non-homogeneous environment of a bacterial cell is still limited. Here, we investigated the mobility of a large set of proteins in the cytoplasm of Escherichia coli, by employing fluorescence correlation spectroscopy (FCS) combined with simulations and theoretical modeling. We conclude that cytoplasmic protein mobility could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. We observed similar size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli. For the faster-diffusing proteins, this size dependence is well consistent with the Stokes-Einstein relation once taking into account the specific dumbbell shape of protein fusions. Pronounced subdiffusion and hindered mobility are only observed for proteins with extensive interactions within the cytoplasm. Finally, while protein diffusion becomes markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity, all of these perturbations affect proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity.

Different Intermolecular Interactions Drive Nonpathogenic Liquid-Liquid Phase Separation and Potentially Pathogenic Fibril Formation by TDP-43.

The liquid-liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, and is critical in the control of many biological processes by forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to de-mix in solution, forming the protein condensed droplets, and amyloid aggregates would form from the droplets after incubation. The molecular interactions regulating TDP-43 LCD LLPS were investigated at the protein fusion equilibrium stage, when the droplets stopped growing after incubation. We found the molecules in the droplet were still liquid-like, but with enhanced intermolecular helix-helix interactions. The protein would only start to aggregate after a lag time and aggregate slower than at the condition when the protein does not phase separately into the droplets, or the molecules have a reduced intermolecular helix-helix interaction. In the protein condensed droplets, a structural transition intermediate toward protein aggregation was discovered involving a decrease in the intermolecular helix-helix interaction and a reduction in the helicity. Our results therefore indicate that different intermolecular interactions drive LLPS and fibril formation. The discovery that TDP-43 LCD aggregation was faster through the pathway without the first protein phase separation supports that LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.

Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin.

The satisfactory visualization of cytoskeletal components in the brain is challenging. The ubiquitous distribution of the networks of microtubules, microfilaments, and intermediate filaments in all the neural tissues, together with the variability in the outcomes of fluorescent protein fusion strategies and their limited applicability to dynamic studies of antibodies and drugs as chromophore vehicles, make classical optical approaches not as effective as for other proteins. When tubulin needs to be studied, the label-free generation of second harmonics is a very suitable option due to the non-centrosymmetric organization of the molecule. This technique, when conjugated to microscopy, can qualitatively describe the volumetric distribution of parallel bundles of microtubules in biological samples, with the additional advantage of working with fresh tissues that are unfixed and unpermeabilized. This work describes how to image tubulin with a commercial second harmonic generation microscopy setup to highlight microtubules in the tubulin-enriched structures of the oligodendrocytes, asin hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) tubulinopathy, a recently described myelin disorder.

Protein fluorescent labeling in live yeast cells using scFv-based probes

The fusion of fluorescent proteins (FPs) to endogenous proteins is a widespread approach for microscopic examination of protein function, expression, and localization in the cell. However, proteins that are sensitive to FP fusion or expressed at low levels are difficult to monitor using this approach. Here, we develop a single-chain fragment variable (scFv)-FP approach to efficiently label Saccharomyces cerevisiae proteins that are tagged with repeats of hemagglutinin (HA)-tag sequences. We demonstrate the successful labeling of DNA-binding proteins and proteins localized to different cellular organelles including the nuclear membrane, peroxisome, Golgi apparatus, and mitochondria. This approach can lead to a significant increase in fluorescence intensity of the labeled protein, allows C'-terminal labeling of difficult-to-tag proteins and increased detection sensitivity of DNA-damage foci. Overall, the development of a scFv-FP labeling approach in yeast provides a general and simple tool for the function and localization analysis of the yeast proteome.

Specific Binding and Endocytosis of Liposomes to HEK293T Cells via Myrisoylated Pre-S1 Peptide Bound to Sodium Taurocholate Cotransporting Polypeptide.

(1) Background: Sodium taurocholate cotransporting polypeptide (NTCP) functions as a key receptor for the hepatitis B virus (HBV) infection. Analyzing HBV and NTCP interaction is an important issue not only for basic research but also for the development of anti-HBV therapeutics. We developed here a novel model system to analyze the interaction of NTCP with liposomes instead of HBV. (2) Methods: Liposomal binding and endocytosis through NTCP in HEK293T cells were achieved by serial treatments of HEL293T cells transiently expressing NTCP-green fluorescence protein (GFP) fusion protein with a synthetic biotinylated pre-S1 peptide (Myr47-Bio) and streptavidin (SA) complex (i.e., Myr47-Bio+SA) followed by biotinylated liposomes. By this procedure, binding of [biotinylated liposomes]-[Myr47-Bio+SA]-[NTCP-GFP] was formed. (3) Results: Using this model system, we found that liposomal binding to NTCP on the cell surface via Myr47-Bio+SA was far more efficient than that to scavenger receptor class B type 1 (SR-B1). Furthermore, liposomes bound to cell surface NTCP via Myr47-Bio+SA were endocytosed into cells after cells were cultured at 37 °C. However, this endocytosis was suppressed by 4 °C or cytochalasin B treatment. (4) Conclusions: This model system will be useful for not only analyzing HBV entry mechanisms but also screening substances to prevent HBV infection.

Candidate Effector Proteins from the Maize Tar Spot Pathogen Phyllachora maydis Localize to Diverse Plant Cell Compartments.

Most fungal pathogens secrete effector proteins into host cells to modulate their immune responses, thereby promoting pathogenesis and fungal growth. One such fungal pathogen is the ascomycete Phyllachora maydis, which causes tar spot disease on leaves of maize (Zea mays). Sequencing of the P. maydis genome revealed 462 putatively secreted proteins, of which 40 contain expected effector-like sequence characteristics. However, the subcellular compartments targeted by P. maydis effector candidate (PmEC) proteins remain unknown, and it will be important to prioritize them for further functional characterization. To test the hypothesis that PmECs target diverse subcellular compartments, cellular locations of super yellow fluorescent protein-tagged PmEC proteins were identified using a Nicotiana benthamiana-based heterologous expression system. Immunoblot analyses showed that most of the PmEC-fluorescent protein fusions accumulated protein in N. benthamiana, indicating that the candidate effectors could be expressed in dicot leaf cells. Laser-scanning confocal microscopy of N. benthamiana epidermal cells revealed that most of the P. maydis putative effectors localized to the nucleus and cytosol. One candidate effector, PmEC01597, localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane, whereas an additional putative effector, PmEC03792, preferentially labelled both the nucleus and nucleolus. Intriguingly, one candidate effector, PmEC04573, consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest that effector candidate proteins from P. maydis target diverse cellular organelles and could thus provide valuable insights into their putative functions, as well as host processes potentially manipulated by this fungal pathogen.

Factor VIII and Von Willebrand Factor Inhibit Osteoclastogenesis in a Humanized Cell Culture Model

Background: Patients with hemophilia A suffer from decreased bone mineral density. It is unknown whether the absence of factor VIII (FVIII) might play a direct causative role. Aim: To investigate the effect of recombinant FVIII (rFVIII, simoctocog alfa) and von Willebrand factor (vWF) on osteoclastogenesis in a humanized two-dimensional (2D) cell culture model. Simoctocog alfa is a rFVIII product manufactured using a human cell line without chemical modification or protein fusion. Methods: CD14+ monocytes from male donors were cultured for 14 days with 10% human serum under the influence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), in the presence and absence of osteoprotegerin (OPG). On days 0, 4, 7 and 11 cells were incubated with 10 IU/ml rFVIII, 10 IU/ml vWF or 10 IU/ml rFVIII/vWF. Tartrate-resistant acid phosphatase (TRAP) activity was measured in the supernatant on day 14 using a colorimetric assay with p-nitrophenylphosphate (pNPP) as substrate. Data were normalized to the condition with mCSF/RANKL alone, or mCSF/RANKL/OPG. Quantitative PCR of cathepsin K (CATK) and nuclear factor of activated T cells (NFATc) was performed. Results: In the absence of OPG, osteoclastogenesis was significantly reduced by the addition of vWF (p<0.001) or the combination rFVIII/VWF (p<0.001). In the presence of OPG, addition of vWF (p<0.001), rFVIII (p<0.05), or the combination rFVIII/VWF (p<0.01) significantly reduced osteoclastogenesis. Similar results were obtained for analyses of CATK and NFATc gene expression. Conclusion: We demonstrated an inhibitory effect of rFVIII, vWF and rFVIII/VWF on osteoclastogenesis in an in vitro model using human serum as opposed to the more commonly used fetal calf serum. As osteoclastogenesis has a high donor-to-donor variation, we are currently testing more donors to see if dependencies on age or blood type exist. These data provide a first insight into mechanisms underlying the reduced bone mass observed in patients with hemophilia A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Design, Heterologous Expression, and Application of an Immobilized Protein Kinase.

Protein kinase A (PKA) is a biologically important enzyme for cell regulation, often referred to as the "central kinase". An immobilized PKA that retains substrate specificity and activity would be a useful tool for laboratory scientists, enabling targeted phosphorylation without interference from downstream kinase contamination or kinase autophosphorylation in sensitive assays. Moreover, it might also provide the benefits of robustness and reusability that are often associated with immobilized enzyme preparations. In this work, we describe the creation of a recombinant PKA fusion protein that incorporates the HaloTag covalent immobilization system. We demonstrate that protein fusion design, including affinity tag placement, is critical for optimal heterologous expression in Escherichia coli. Furthermore, we demonstrate various applications of our immobilized PKA, including the phosphorylation of recombinant PKA substrates, such as vasodilator-stimulated phosphoprotein, and endogenous PKA substrates in a cell lysate. This immobilized PKA also possesses robust activity and reusability over multiple trials. This work holds promise as a generalizable strategy for the production and application of immobilized protein kinases.

Regulation of caries-induced pulp inflammation by NLRP3 inflammasome: A laboratory-based investigation.

To evaluate the expression and function of the nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in caries induced pulpitis. NLRP3 expression was determined with immunohistochemistry in the dental pulp and qPCR in dental pulp cells (DPCs). THP-1 macrophages expressing the apoptosis-related speck-like protein (ASC) and green fluorescent protein (GFP) fusion protein were used to assess NLRP3 inflammasome activation by live cell imaging, following treatment with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Caspase I inhibitor was used to confirm inflammasome activation. An ex-vivo pulpitis model in which the DPCs were co-cultured with THP-1 macrophages was used to study the effect of the NLRP3 inflammasome inhibitor (MCC950), and cytokines were measured using ELISA and multiplex array. Data were analysed using the t-test or anova followed by a Bonferroni post hoc test with the level of significance set at p ≤ .05. NLRP3 inflammasome was differentially expressed in dental pulp of sound and carious teeth. Treatment of DPCs with LTA significantly upregulates NLRP3 and IL-1 β-expression (p < .05) and in induces more ASC specks formation compared to LPS. IL-β release in response to LTA treatment is significantly reduced with Caspase I inhibitor suggesting inflammasome dependent mechanism (p < .01). NLRP3-specific inhibitor, MCC950, significantly reduced IL-1β and IL-6 in an ex-vivo pulpitis model (p < .01) but had no effect on IL-8 or matrix metalloproteinase-9 (MMP-9). Expression and upregulation of NLRP3 inflammasome with caries and LTA treatment suggest a role in caries-induced pulpitis. NLRP3 inhibitor attenuated the release of selective inflammatory cytokines and could be a potential treatment target that merit further investigation.

Ferritin-displayed antigen nanoparticles and nanobody-horseradish peroxidase fusions based-competitive ELISA for the rapid and sensitive detection of antibody against African swine fever virus

African swine fever (ASF) is a highly contagious and lethal hemorrhagic disease, which has brought great distress and economic losses to the world pig industry. For lack of approved vaccines and effective treatment, accurate early detection, both ASF virus (ASFV) antigen and antibody detection in the field, is one of the most important aspects for preventing the outbreak and spread of ASFV. Here we screened the specific nanobodies against the ASFV-P30 protein from the library of immunized Bactrian camel and fused them with horseradish peroxidase (HRP) for expression in HEK293T cells. Moreover, the P30 protein was designed and displayed on the surface of ferritin, the fusion nanocages. Then we developed a novel competitive ELISA (cELISA) based on ferritin-displayed P30 nanoparticles protein (P30-Fn) and nanobody-horseradish peroxidase fusions (P30–Nb71-vHRP) for rapid detection of ASFV antibody in serum sample with high sensitivity and specificity. The cELISA can detect as far as 1:200 diluted clinically positive serums and show no cross-reactivity with positive serums against others porcine viruses, such as PRRSV, PPV, CSFV, TGEV, PEDV, PCV and SIV. The agreement rate is 98.1% with the test results of the commercial ELISA kit for the detection of a total of 267 clinical samples, and the kappa value is 0.96. Furthermore, the cELISA exhibits a good repeatability and the intra- and inter-assay coefficient of variation are less than 10%. In summary, the newly developed cELISA is a simple, rapid, sensitive and low-cost immunoassay, which has a great potential for ASFV antibody detection in clinical samples.

Insight into the molecular structure and function of peptidoglycan recognition protein SC2 (PGRP-SC2) from Amphiprion clarkii: Investigating the role in innate immunity

Peptidoglycan recognition proteins (PGRPs) belong to the pattern recognition receptor (PRR) family and are conserved from insects to mammals. PGRPs show specific binding abilities to peptidoglycans (PGNs) in various microbes. In this study, molecular and functional analyses of PGRP-SC2 from Amphiprion clarkii (AcPGRP-SC2) were conducted. The 492 bp ORF of AcPGRP-SC2 encoded a protein of 164 amino acids with a molecular weight of 17.58 kDa and pI of 8.9. The PGRP superfamily domain was identified from the protein sequence of AcPGRP-SC2 and sequence similarities were observed with homologous proteins. Quantitative polymerase chain reaction (qPCR) analysis revealed that AcPGRP-SC2 transcripts were ubiquitously expressed in all tested tissues, with high levels in the skin, and transcript expression was significantly modulated by immune stimulation with lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (poly I:C), and Vibrio harveyi post-immune challenge. Recombinant AcPGRP-SC2 with the maltose-binding protein fusion (rAcPGRP-SC2) was used to evaluate LPS-, PGN-, and bacterial-binding activities and to conduct bacterial agglutination assays, and the results demonstrated that AcPGRP-SC2 exhibited bacterial recognition, binding, and colonization abilities to a range of Gram-positive and Gram-negative bacterial strains. Moreover, rAcPGRP-SC2-pre-treated Fat Head Minnow (FHM) cells exhibited significant upregulation in NF-ĸB1, NF-ĸB2, and stat3 expression upon treatment with killed bacteria. Taken together, our findings suggest that AcPGRP-SC2 plays an important role in the immune response against microbial pathogens in A. clarkii.

Efficient antigen delivery by dendritic cell-targeting peptide via nucleolin confers superior vaccine effects in mice

SummaryEfficient delivery of subunit vaccines to dendritic cells (DCs) is necessary to improve vaccine efficacy, because the vaccine antigen alone cannot induce sufficient protective immunity. Here, we identified DC-targeting peptides using a phage display system and demonstrated the potential of these peptides as antigen-delivery carriers to improve subunit vaccine effectiveness in mice. The fusion of antigen proteins and peptides with DC-targeting peptides induced strong antigen-specific IgG responses, even in the absence of adjuvants. In addition, the DC-targeting peptide improved the distribution of antigens to DCs and antigen presentation by DCs. The combined use of an adjuvant with a DC-targeting peptide improved the effectiveness of the vaccine. Furthermore, nucleolin, located on the DC surface, was identified as the receptor for DC-targeting peptide, and nucleolin was indispensable for the vaccine effect of the DC-targeting peptide. Overall, the findings of this study could be useful for developing subunit vaccines against infectious diseases.

FRET-FLIM to Determine Protein Interactions and Membrane Topology of Enzyme Complexes.

Determining protein-protein interactions is vital for gaining knowledge on cellular and metabolic processes including enzyme complexes and metabolons. Förster resonance energy transfer with fluorescence lifetime imaging microscopy (FRET-FLIM) is an advanced imaging methodology that allows for the quantitative detection of protein-protein interactions. In this method, proteins of interest for interaction studies are fused to different fluorophores such as enhanced green fluorescent protein (eGFP; donor molecule) and monomeric red fluorescent protein (mRFP; acceptor molecule). Energy transfer between the two fluorophore groups can only occur efficiently when the proteins of interest are in close physical proximity, around ≤10 nm, and therefore are most likely interacting. FRET-FLIM measures the decrease in excited-state lifetime of the donor fluorophore (eGFP) with and without the presence of the acceptor (mRFP) and can therefore give information on protein-protein interactions and the membrane topology of the tested protein. Here we describe the production of fluorescent protein fusions for FRET-FLIM analysis in tobacco leaf epidermal cells using Agrobacterium-mediated plant transformation and a FRET-FLIM data acquisition and analysis protocol in plant cells. These protocols are applicable and can be adapted for both membrane and soluble proteins in different cellular localizations. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protein expression in tobacco leaf cells via transient Agrobacterium-mediated plant transformation Basic Protocol 2: FRET-FLIM data acquisition and analysis.

Flagellin from Salmonella enteritidis Enhances the Immune Response of Fused F18 from Enterotoxigenic Escherichia coli.

F18 plays an important role in helping Enterotoxigenic Escherichia coli (ETEC) binds to specific receptors on small intestinal enterocytes, followed by secreting of toxins causing diarrhea in post-weaning piglets (post-weaning diarrhea, PWD). However, the F18 subunit vaccine is not sufficient to stimulate an immune response that can protect weaning pigs from F18-positive ETEC (F18+ETEC). Recently, a body of evidence shows that flagellin protein (FliC) helps to increase the immunity of fused proteins. Therefore, in this study, we combined FliC with F18 to enhance the immune response of F18. The f18 gene was obtained from F18+ETEC, then was fused with the fliC gene. The expression of recombinant FliC-F18 protein was induced by Isopropyl-beta-D-Thiogalactopyranoside (IPTG). The purified protein was tested in vivo in mouse models to evaluate the immunostimulation. Results showed that the fusion of FliC and F18 protein increased the production of anti-F18 antibodies. Besides, the anti-F18 antibody in the collected antiserum specifically identified F18+ETEC. This result provides proof-of-concept for the development of subunit vaccine to prevent PWD using F18 antigen.

Diversity and Abundance of Soil Collembola during GM Rice Overexpressing Cry1B-Cry1Aa Cultivations at Four Confined Field Trials in West Java.

Collembola (springtails) is an important soil biology indicator to monitor toxicity or ecological disturbances in the ecosystem. The impact of Bacillus thuringiensis (Bt) rice cv Rojolele events expressing Cry1B-Cry1Aa driven by the maize ubiquitin promoter resistant to yellow rice stem borer (YSB, Scirpophaga incertulas Walker) on non-target Collembola community was assessed. The experiment was performed at four locations under confined field trials according to the Indonesia's environmental safety regulation on genetically engineered crops. Six transgenic rice events were tested with non-transgenic Rojolele and the moderately resistant IR42 rice varieties as controls. The experimental design was randomised block design with three replicates. Collembola were collected from the bunds between plots using pitfall and Berlese funnel traps at seedling, vegetative and generative stages, as well as at harvesting time. The results showed that Collembola abundance and diversity were significantly affected by both experimental sites and observation times. However, no significant differences in Collembola diversity and abundance between Bt rice and non-Bt controls were observed. Thus, we can conclude that the cultivation of the Bt rice cv Rojolele events expressing Cry1B-Cry1Aa protein fusion do not adversely affect biodiversity and abundance of Collembola at the four confined rice fields.

Recurrent but Short-Lived Duplications of Centromeric Proteins in Holocentric Caenorhabditis Species.

Centromeric histones (CenH3s) are essential for chromosome inheritance during cell division in most eukaryotes. CenH3 genes have rapidly evolved and undergone repeated gene duplications and diversification in many plant and animal species. In Caenorhabditis species, two independent duplications of CenH3 (named hcp-3 for HoloCentric chromosome-binding Protein 3) were previously identified in C. elegans and C. remanei. Using phylogenomic analyses in 32 Caenorhabditis species, we find strict retention of the ancestral hcp-3 gene and 10 independent duplications. Most hcp-3L (hcp-3-like) paralogs are only found in 1–2 species, are expressed in both males and females/hermaphrodites, and encode histone fold domains with 69–100% identity to ancestral hcp-3. We identified novel N-terminal protein motifs, including putative kinetochore protein-interacting motifs and a potential separase cleavage site, which are well conserved across Caenorhabditis HCP-3 proteins. Other N-terminal motifs vary in their retention across paralogs or species, revealing potential subfunctionalization or functional loss following duplication. An N-terminal extension in the hcp-3L gene of C. afra revealed an unprecedented protein fusion, where hcp-3L fused to duplicated segments from hcp-4 (nematode CENP-C). By extending our analyses beyond CenH3, we found gene duplications of six inner and outer kinetochore genes in Caenorhabditis, which appear to have been retained independent of hcp-3 duplications. Our findings suggest that centromeric protein duplications occur frequently in Caenorhabditis nematodes, are selectively retained for short evolutionary periods, then degenerate or are lost entirely. We hypothesize that unique challenges associated with holocentricity in Caenorhabditis may lead to this rapid “revolving door” of kinetochore protein paralogs.

Green fluorescent protein and phase contrast image fusion via Spectral TV filter-based decomposition

The spectral filter is one of the crucial tools for signal scale and texture analysis. The main function is to decompose a raw image into a series of multi-scale signatures in the spectral domain. For cross-modal fusion, most of the existing approaches naively employ the decomposition of spectral features to achieve image reconstruction. Unfortunately, it does not sufficiently explore the potential cues of different level components in the spatial domain. In this work, we describe an effective decomposition approach using the spectral filter. Specifically, we aim to build a multi-scale image decomposition framework from the spectral domain to the spatial domain, which is called the multi-scale spatial decomposition approach (MSD). Moreover, building upon spectral total variation (TV), we characterize the spectral signatures into various spatial levels with different filters. A fusion scheme of green fluorescent protein and phase contrast image is introduced, leveraging the multi-scale spatial decomposition approach. The source image is first separated by applying MSD into three spatial bands, namely high-frequency bands, low-frequency bands and structure bands. Our method is simple and effective. The divided bands are obtained by a set of filters and could be processed with the specific fusion strategies to extract the features of the source image. In addition, experiments show that our method produces promising results than other current popular methods.

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.