Signal Peptide Sequence Research Articles

Genome-wide high-throughput signal peptide screening via plasmid pUC256E improves protease secretion in Lactiplantibacillus plantarum and Pediococcus acidilactici

BackgroundProteases catalyze the hydrolysis of peptide bonds of proteins, thereby improving dietary protein digestibility, nutrient availability, as well as flavor and texture of fermented food and feed products. The lactobacilli Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) and Pediococcus acidilactici are widely used in food and feed fermentations due to their broad metabolic capabilities and safe use. However, extracellular protease activity in these two species is low. Here, we optimized protease expression and secretion in L. plantarum and P. acidilactici via a genetic engineering strategy.ResultsTo this end, we first developed a versatile and stable plasmid, pUC256E, which can propagate in both L. plantarum and P. acidilactici. We then confirmed expression and secretion of protease PepG1 as a functional enzyme in both strains with the aid of the previously described L. plantarum-derived signal peptide LP_0373. To further increase secretion of PepG1, we carried out a genome-wide experimental screening of signal peptide functionality. A total of 155 predicted signal peptides originating from L. plantarum and 110 predicted signal peptides from P. acidilactici were expressed and screened for extracellular proteolytic activity in the two different strains, respectively. We identified 12 L. plantarum signal peptides and eight P. acidilactici signal peptides that resulted in improved yield of secreted PepG1. No significant correlation was found between signal peptide sequence properties and its performance with PepG1.ConclusionThe vector developed here provides a powerful tool for rapid experimental screening of signal peptides in both L. plantarum and P. acidilactici. Moreover, the set of novel signal peptides identified was widely distributed across strains of the same species and even across some closely related species. This indicates their potential applicability also for the secretion of other proteins of interest in other L. plantarum or P. acidilactici host strains. Our findings demonstrate that screening a library of homologous signal peptides is an attractive strategy to identify the optimal signal peptide for the target protein, resulting in improved protein export.

The genetic variations in CSN2 gene of Indian sheep breeds affect its protein stability and function

Milk from minor dairy animals, such as sheep and goats, is gaining popularity, and its quality is viewed as significant in light of current healthy dietary trends. The β-casein (coded by CSN2 gene) is the most abundant casein protein family member accounting for more than half of the total casein protein in sheep milk. The goal of this work was to find genetic polymorphism (SNP) in the sheep CSN2 gene and predict how SNPs will affect protein stability and function. The mRNA was isolated from milk somatic cells of nine sheep breeds from northern India to get the complete coding β-casein gene. The exon seven of β-casein was screened for the genetic variations at DNA level by PCR-SSCP method, from 300 sheep belonging to nine breeds. The comparative sequence analysis in Indian sheep breeds revealed 21 novel CSN2 protein variants, maximum of the variants raised due to the SNPs in the exon seven of the gene. The computational analysis of deduced amino acids substitutions in CSN2 protein demonstrated that these changes in the mature protein could change the structure, stability and function of the milk protein. Variations in the signal peptide and mature protein sequences were projected to have a negative impact on the casein protein's stability and function. These changes were also projected to disrupt CSN2 protein post-translational modifications, which could affect different technical aspects of milk, thereby jeopardizing crucial milk coagulation features like rennet coagulation time and curd hardness.

Copper toxicity towards Campylobacter jejuni is enhanced by the nickel chelator dimethylglyoxime.

The nickel (Ni)-chelator dimethylglyoxime (DMG) was found to be bacteriostatic towards Campylobacter jejuni. Supplementation of nickel to DMG-containing media restored bacterial growth, whereas supplementation of cobalt or zinc had no effect on the growth inhibition. Unexpectedly, the combination of millimolar levels of DMG with micromolar levels of copper (Cu) was bactericidal, an effect not seen in select Gram-negative pathogenic bacteria. Both the cytoplasmic Ni-binding chaperone SlyD and the twin arginine translocation (Tat)-dependent periplasmic copper oxidase CueO were found to play a central role in the Cu-DMG hypersensitivity phenotype. Ni-replete SlyD is needed for Tat-dependent CueO translocation to the periplasm, whereas Ni-depleted (DMG-treated) SlyD is unable to interact with the CueO Tat signal peptide, leading to mislocalization of CueO and increased copper sensitivity. In support of this model, C. jejuni ΔslyD and ΔcueO mutants were more sensitive to copper than the wild-type (WT); CueO was less abundant in the periplasmic fraction of ΔslyD or DMG-grown WT cells, compared to WT cells grown on plain medium; SlyD binds the CueO signal sequence peptide, with DMG inhibiting and nickel enhancing the binding, respectively. Injection of Cu-DMG into Galleria mellonella before C. jejuni inoculation significantly increased the insect survival rate compared to the control group. In chickens, oral administration of DMG or Cu-DMG decreased and even abolished C. jejuni colonization in some cases, compared to both water-only and Cu-only control groups. The latter finding is important, since campylobacteriosis is the leading bacterial foodborne infection, and chicken meat constitutes the major foodborne source.

Biogenesis of hepatitis B virus e antigen is driven by translocon-associated protein complex and regulated by conserved cysteine residues within its signal peptide sequence.

Hepatitis B virus uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro‐translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon‐associated protein complex. We have found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we have identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool.

Cloning, expression, and function of ferritins in the tick Haemaphysalis flava

The full-length cDNA of two ferritins of Haemaphysalis flava were cloned after which recombinant Hf-FER1 and Hf-FER2 were expressed and their function was analyzed. In addition, RNA interference (RNAi) based on the injection of Hf-fer1 or Hf-fer2 dsRNA into fully engorged female ticks was performed. The cDNA encoding Hf-FER1 is 834 bp in length. It contains an iron-responsive element in the 5ʹ untranslated region and encodes 174 amino acid residues. The full-length cDNA of Hf-FER2 contains 696 bp and encodes 199 amino acids, including a putative signal peptide sequence. Hf-FER1 and Hf-FER2 both have the ferroxidase iron center and the ferrihydrite nucleation center. The evolutionary relationship of Hf-FER1 and Hf-FER2 was established, and the predicted quaternary structures were assembled as typical spherical shells composed of 24 subunits which was demonstrated by nature PAGE. Real-time PCR showed that Hf-fer1 and Hf-fer2 were expressed in all developmental stages, with the highest expression in fully engorged females. The expression of Hf-fer1 and Hf-fer2 were relatively high in unfed larvae. Hf-fer1 was expressed in all tissues and was especially abundant in the salivary glands of fully engorged females. In contrast, the highest levels of Hf-fer2 were found in the midgut of fully engorged females, and no expression was found in the salivary glands of this life stage. Both recombinant Hf-FER1 and Hf-FER2 had iron-binding capabilities. Silencing of both Hf-fer1 and Hf-fer2 affected fecundity. Compared to the control, the percentage of ticks that laid eggs in the Hf-fer1 and Hf-fer2 RNAi groups was 73.3% and 66.7%, respectively. The silenced ticks that laid eggs had lower egg weight to body weight ratios, and the eggs had abnormal morphologies. The hatchability of eggs with normal morphology in the Hf-fer1 and Hf-fer2 silenced groups was 47.8% and 22.8%, respectively, which was significantly different from the control group (P < 0.005). These findings indicate that Hf-FER1 and Hf-FER2 play important roles in the iron storage of H. flava.

Evolution of aldehyde dehydrogenase genes and proteins in diploid and allotetraploid Xenopus frog species

At least 19 human aldehyde dehydrogenase (ALDH) genes and enzymes have been studied among vertebrate organisms. BLAT and BLAST analyses were undertaken of Xenopus tropicalis (western clawed frog) and Xenopus laevis (African clawed frog) genomes which are related diploid (N = 20) and allotetraploid (N = 36) species, respectively. The corresponding ALDH genes and proteins within these Xenopus genomes were identified and studied. Evidence is presented for tetraploid copies of 10 Xenopus laevis ALDH genes, whereas another 7 identified ALDH genes were diploid in nature. Xenopus laevis and Xenopus tropicalis ALDH amino acid sequences were highly homologous with the human enzymes, with the exception of the mitochondrial signal peptide sequences. Amino acids performing catalytic and structural roles were conserved and identified based on previous reports of 3D structures for the corresponding mammalian enzymes.

Expression of the Bacillus subtilis TasA signal peptide leads to cell death in Escherichia coli due to inefficient cleavage by LepB

Bacillus subtilis has five type I signal peptidases, one of these, SipW, is an archaeal-like peptidase. SipW is expressed in an operon (tapA-sipW-tasA) and is responsible for removing the signal peptide from two proteins: TapA and TasA. It is unclear from the signal peptide sequence of TasA and TapA, why an archaeal-like signal peptidase is required for their processing. Bioinformatic analysis of TasA and TapA indicates that both contain highly similar signal peptide cleavage sites, both predicted to be cleaved by Escherichia coli signal peptidase I, LepB. We show that expressing full length TasA in E. coli is toxic and leads to cell death. To determine if this phenotype is due to the inability of the E. coli LepB to process the TasA signal peptide, we fused the TasA signal peptide and two amino acids of mature TasA (up to P2′) to both maltose binding protein (MBP) and β-lactamase (Bla). We observed a defect in secretion, indicated by an abundance of unprocessed protein with both TasA-MBP and TasA-Bla fusions. A series of mutations in both TasA-MBP and TasA-Bla were made around the junction of the TasA signal peptide and the fusion protein. Both of these studies indicate that residues around the predicted TasA signal sequence cleavage site, particularly the sequence from P3 to P2′, inhibit processing by LepB. The cell death observed when TasA and TasA signal sequence fusion proteins are expressed is likely due to the TasA signal peptide blocking LepB and thereby the general secretion pathway.

Structure prediction and biological activity analysis of dybowskin-1ST antimicrobial peptide in Rana dybowskii

The evolution, structure and antigenic epitopes prediction of Rana dybowskii antimicrobial peptide dybowskin-1ST were carried out using bioinformatics software available online. Its antibacterial mechanism and structural properties were analyzed, and its activity was verified by applying wound healing assay in mice and bacteriostatic assay in vitro. This provides the theoretical basis for the improvement of parental peptide and the development of novel derivative peptides. The software MEGA_X were used to conduct homology alignment and to construct a phylogenetic tree. The online software ProtParam, ProtScale, PeptideCutter, signal, TMHMM Server were respectively used to predict the physicochemical parameters, hydrophilia/hydrophobicity, shear sites, signal peptides, and transmembrane domains of dybowskin-1ST. The online software SOPMA, Jpred4, DNAstar Protean were used to predict the secondary structure of dybowskin-1ST, and SWISS-MODEL, I-TASSER were used to predict the tertiary structure. ABCpred and SYFPEITHI were respectively used to predict its B-and T-cell epitopes. The effect of dybowskin-1ST on the wound healing was observed on experimental mice. Kirby-Bauer method and dilution method were used to determine the bacteriostatic activity of dybowskin-1ST. The dybowskin-1ST consists of 59 amino acid residues, of which leucine accounts for 16.9%, with a molecular formula of C₃₁₈H₅₁₀N₈₀O₉₃S₂. Its theoretical isoelectric point is 5.10 and the charge is -2. The dybowskin-1ST and dybowskin-1CDYa are closely related phylogenetically. The secondary structure of dybowskin-1ST predicted by the three methods were similar, which consisted of α-helix (44.07%), extended strand (16.95%), β-turns (3.39%), and random coil (35.39%). The prediction of tertiary structure showed that dybowskin-1ST was mainly composed of α-helix, and it was regarded as a hydrophilic protein with signal peptide sequence. Subcellular localization analysis showed that the probability of secreting the mitochondrial targeted peptides was 0.944. Dybowskin-1ST is an extracellular protein with no transmembrane structure region, but contains seven phosphorylation sites, three T-cell epitopes and eight B-cell epitopes. The dybowskin-1ST promoted wound healing and effectively inhibited the growth of Escherichia coli and Staphylococcus aureus. However, it had limited antibacterial activity against fungi and drug-resistant bacteria. Although the structure of dybowskin-1ST is rich in α-helix, the verification experiments showed that its antibacterial ability needs to be enhanced. The reason may be that it is a negatively charged and hydrophilic protein, and amino acid modification with the aim of increasing the number of positive charges and changing the hydrophobicity may be used to obtain derived peptides with enhanced activity.

Refining the N-Termini of the SARS-CoV-2 Spike Protein and Its Discrete Receptor-Binding Domain.

Previous work employing five SARS-CoV-2 spike protein receptor-binding domain (RBD) constructs, comprising versions originally developed by Mt. Sinai or the Ragon Institute and later optimized in-house, revealed potential heterogeneity which led to questions regarding variable seropositivity assay performance. Each construct was subjected to N-deglycosylation and subsequent intact mass analysis, revealing significant deviations from predicted theoretical mass for all five proteins. Complementary tandem MS/MS analysis revealed the presence of an additional pyroGlu residue on the N-termini of the two Mt. Sinai RBD constructs, as well as on the N-terminus of the full-length spike protein from which they were derived, thus explaining the observed mass shift and definitively establishing the spike protein N-terminal sequence. Moreover, the observed mass additions for the three Ragon Institute RBD constructs were identified as variable N-terminal cleavage points within the signal peptide sequence employed for recombinant expression. To resolve this issue and minimize heterogeneity for further seropositivity assay development, the best-performing RBD construct was further optimized to exhibit complete homogeneity, as determined by both intact mass and tandem MS/MS analysis. This new RBD construct has been validated for seropositivity assay performance, is available to the greater scientific community, and is recommended for use in future assay development.

Intracellular protein delivery using QRPL – A vacuolar targeting signal on carboxypeptidase Y

The signal peptide sequence is known to increase transport efficiency to organelles in eukaryotic cells. In this study, we focus on the signal peptide of the vacuolar protein for vacuolar targeting. The signal peptide sequence QRPL of carboxypeptidase Y (CPY) was inserted inside the interest protein that does not locate in the vacuole for vacuolar targeting. We constructed recombinant strains MBTL-Q-DJ1 and MBTL-Q-DJ2 containing QRPL and green florescent protein (GFP) or aldehyde dehydrogenase 6 (ALD6), respectively. The protein location was then confirmed by confocal microscopy. Fascinatingly, the green fluorescent protein that contains QRPL inside the sequence could be expressed faster than its natural form (within 1 h after induction). Also, the aldehyde removal activity of ALD6 protein in the recombinant yeast was then analyzed by measuring the luminescent intensity in Vibrio fischeri. We confirmed that MBTL-Q-DJ2 containing ALD6 protein has the aldehydes-reducing ability, and in particular, the highest efficiency showed at 500 μg/μL of vacuolar enzyme. In summary, the signal peptide QRPL could be used not only to transport proteins accurately to vacuole but also to improve the protein activity and shorten the induction time.

Comparative Genomics of Clostridium perfringens Reveals Patterns of Host-Associated Phylogenetic Clades and Virulence Factors.

Clostridium perfringens is an opportunistic pathogenic bacterium that infects both animals and humans. Clostridium perfringens genomes encode a diverse array of toxins and virulence proteins, which continues to expand as more genomes are sequenced. In this study, the genomes of 44 C. perfringens strains isolated from intestinal sections of diseased cattle and from broiler chickens from diseased and healthy flocks were sequenced. These newly assembled genomes were compared to 141 publicly available C. perfringens genome assemblies, by aligning known toxin and virulence protein sequences in the assemblies using BLASTp. The genes for alpha toxin, collagenase, a sialidase (nanH), and alpha-clostripain were present in at least 99% of assemblies analyzed. In contrast, beta toxin, epsilon toxin, iota toxin, and binary enterotoxin of toxinotypes B, C, D, and E were present in less than 5% of assemblies analyzed. Additional sequence variants of beta2 toxin were detected, some of which were missing the leader or signal peptide sequences and therefore likely not secreted. Some pore-forming toxins involved in intestinal diseases were host-associated, the netB gene was only found in avian isolates, while netE, netF, and netG were only present in canine and equine isolates. Alveolysin was positively associated with canine and equine strains and only present in a single monophyletic clade. Strains from ruminant were not associated with known virulence factors and, except for the food poisoning associated clade, were present across the phylogenetic diversity identified to date for C. perfringens. Many C. perfringens strains associated with food poisoning lacked the genes for hyaluronidases and sialidases, important for attaching to and digesting complex carbohydrates found in animal tissues. Overall, the diversity of virulence factors in C. perfringens makes these species capable of causing disease in a wide variety of hosts and niches.

Association of KCTD15 gene with fat deposition in pigs.

KCTD15 is associated with body mass index and fat deposition in humans, mice and chickens. However, the function of KCTD15 in pig fat deposition remains unclear. In this study, we cloned and analysed the cDNA sequence of porcine KCTD15. The full length of the mRNA sequence of KCTD15 is 4,091bp, encoding 283 amino acids. The protein is hydrophilic, it has a relative molecular mass of about 31.9kDa and an isoelectric point of 7.09 with no signal peptide sequence or transmembrane structure. Expression analysis showed that KCTD15 expression level was significantly higher in the tissues of Large White pigs (LW) than in those of Tibetan pigs (TP) and Diannan Small-ear pigs (DN) at 6months of age, whereas its expression level in embryonic tissues of LW at 60days was lower than that in tissues of TP and Wujin pigs (WJ). In pig primary adipocytes, the expression level of KCTD15 is high in the early stage of differentiation and gradually decreases in later stages. Additionally, the single-nucleotide polymorphism (SNP) site T-2030C (T/C mutation, located 2,030bp upstream of the start codon) showed a dominant allele T with high promoter activity in the LW population and a dominant allele C in the TP and WJ populations. Our results indicate that KCTD15 is involved in pig fat deposition and that T-2030C is an important regulatory site for transcriptional activity, affecting fat deposition.

Efficient Secretion of Murine IL-2 From an Attenuated Strain of Clostridium sporogenes, a Novel Delivery Vehicle for Cancer Immunotherapy.

Despite a history dating back to the 1800s, using Clostridium bacteria to treat cancer has not advanced beyond the observation that they can colonise and partially destroy solid tumours. Progress has been hampered by their inability to eradicate the viable portion of tumours, and an instinctive anxiety around injecting patients with a bacterium whose close relatives cause tetanus and botulism. However, recent advances in techniques to genetically engineer Clostridium species gives cause to revisit this concept. This paper illustrates these developments through the attenuation of C. sporogenes to enhance its clinical safety, and through the expression and secretion of an immunotherapeutic. An 8.6 kb sequence, corresponding to a haemolysin operon, was deleted from the genome and replaced with a short non-coding sequence. The resultant phenotype of this strain, named C. sporogenes-NT, showed a reduction of haemolysis to levels similar to the probiotic strain, C. butyricum M588. Comparison to the parental strain showed no change in growth or sporulation. Following injection of tumour-bearing mice with purified spores of the attenuated strain, high levels of germination were detected in all tumours. Very low levels of spores and vegetative cells were detected in the spleen and lymph nodes. The new strain was transformed with four different murine IL-2-expressing plasmids, differentiated by promoter and signal peptide sequences. Biologically active mIL-2, recovered from the extracellular fraction of bacterial cultures, was shown to stimulate proliferation of T cells. With this investigation we propose a new, safer candidate for intratumoral delivery of cancer immunotherapeutics.

Cathepsin L gene: Molecular characterization, functional analysis and expression profile in the endometrium of goat (Capra hircus)

In ruminant species, endometrial remodeling plays a pivotal role in regulation of estrous cycle and establishment of pregnancy. Among interferon tau (IFNT) stimulated genes, cathepsin (CTS) L is one of them, which is up-regulated in the endometrium during early pregnancy and plays an important role in endometrial remodeling and embryo development. However, the specific role of CTSL is not yet documented in goats. Therefore, in the present study, coding sequence (CDS) of CTSL was cloned, characterized, and its temporal expression profile was also examined in the endometrium of caprine (cp) for the first time. A fragment of cpCTSL, 963 bp in length, was amplified from complementary DNA (cDNA) with a 936 bp coding sequence, which encodes a polypeptide of 312 amino acids. Coding and deduced amino acid sequences of cpCTSL exhibited 72.8 to 99.3% identities with other species. A polypeptide of 312 amino acids of cpCTSL revealed three domains, a signal peptide sequence, a pro-domain and a partially mature domain. Further, the relative expression of cpCTSL mRNA and protein was determined by quantitative real-time PCR and western blot, respectively, in the endometrium of pregnant and cyclic does. Both cpCTSL mRNA and protein were expressed maximally (P < 0.05) during early pregnancy and these were 11.34 and 5.48-fold greater (P < 0.05), respectively, as compared to cyclic does. It is concluded that CTSL is mostly similar in structure to other species and increased level in the endometrium indicates its involvement in endometrial remodeling to sustain pregnancy in goats.

Functional analysis of the orphan genes Tssor-3 and Tssor-4 in male Plutella xylostella

Orphan genes are genes with no sequence homologues in other species. Here, we identified two orphan genes, namely, Tssor-3 and Tssor-4, in Plutella xylostella. Both genes contained a signal peptide sequence, suggesting their functions as secreted proteins. Expression pattern analysis based on real-time quantitative PCR (qPCR) showed that both orphan genes were specifically expressed in all male gonads except the testes. The expression of both the orphan genes peaked at the male adult stage. Immunofluorescence assays suggested that the two proteins were seminal proteins, indicating their potential roles in male reproductive regulation. To further explain their functions, we knocked down the expression of these two genes by RNA interference (RNAi). The results showed that the expression of Tssor-3 and Tssor-4 was significantly downregulated at 24 h after injection compared to that of the controls. Biological assays showed that the number of laid eggs and the hatching rate of offspring eggs were significantly reduced when the expression of Tssor-3 and Tssor-4 was reduced, suggesting that the two orphan genes played a role in male fertility in P. xylostella. Our results provide evidence that orphan genes are involved in male reproductive regulation, which is important for male fitness during evolution.

Coated recombinant Escherichia coli for delayed release of β-mannanase in the water-based fracturing fluid

A low-temperature active glycosyl hydrolase family 26(GH26) β-mannanase (Man37) was identified in Bacillus velezensis BS-37 by gene mining. The β-mannanase Man37 contained a signal peptide at the N-terminus. The Man37 and Man37△1−25 (without the signal peptide sequence) coding sequences were successfully expressed in Escherichia coli BL21(DE3) using the pET-28a(+) vector. The specific activity of the purified Man37 and Man37△1−25 with guar gum as substrate was 98.12 and 150.34 U/mg, respectively. Man37△1−25 was superior to Man37 due to its higher specific activity and soluble expression. The temperature and pH range of Man37△1−25 was 20−55 °C and pH5.0–10.0, respectively. The enzyme could effectively reduce the viscosity of fracturing fluid from 6200 to 1000 mPa·s after 10 min at 25 °C, which demonstrates its application potential as a gel-breaker in low-temperature hydrocarbon reservoirs. Uncoated recombinant Man37△1−25 cells could completely reduce the viscosity of water-based fracturing fluid within only 3 h, while was greatly prolonged to 18 h with coating polyethylenimine-glutaraldehyde (PEI-GA). Thus, this study provides an effective solution for premature degradation caused by the addition of water-based fracturing fluid together with a short-acting gel-breaker.

Variable posttranslational modifications of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.

A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection.

Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes’ expression are located in the promoter and 3’-untranslated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1–10 bp), but we detected several larger deletions as well (100–300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40–80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression.

Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts

Key messageModified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful.Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

ZFN-mediated in vivo gene editing in hepatocytes leads to supraphysiologic α-Gal A activity and effective substrate reduction in Fabry mice

Fabry disease, a lysosomal storage disorder resulting from thedeficient activity of α-galactosidase A (α-Gal A), is characterized by cardiac, renal, and/or cerebrovascular disease due to progressive accumulation of the enzyme's substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). We report here the preclinical evaluation of liver-targeted invivo genome editing using zinc-finger nuclease (ZFN) technology to insert the human α-galactosidase A (hGLA) cDNA into the albumin "safe harbor" locus of Fabry mice, thereby generating an albumin-α-Gal A fusion protein. The mature α-Gal A protein is secreted into the circulation for subsequent mannose-6-phosphate receptor-mediated tissue uptake. Donor vector optimization studies showed that replacing the hGLA cDNA signal peptide sequence with that of human iduronate 2-sulfatase (IDS) achieved higher transgene expression. Intravenous adeno-associated virus (AAV) 2/8-mediated co-delivery of the IDS-hGLA donor and ZFNs targeting the albumin locus resulted in continuous, supraphysiological plasma and tissue α-Gal A activities, which essentially normalized Gb3 and Lyso-Gb3 levels in key tissues of pathology. Notably, this was achieved with <10% of hepatocytes being edited to express hGLA, occurring mostly via non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). These studies indicate that ZFN-mediated invivo genome editing has the potential to be an effective one-time therapy for Fabry disease.