Secretion Signal Peptide Research Articles

Clonorchis sinensis secretory protein CsAg17 vaccine induces immune protection

BackgroundClonorchiasis is endemic in East and Southeast Asian countries. For a preventive strategy against infectious diseases, vaccination is the most effective. Here, we evaluated the molecular characteristics and immune responses of CsAg17 protein from Clonorchis sinensis, and investigated its protective effects against C. sinensis challenge.MethodsA cDNA clone encoding CsAg17 protein and containing a secretory signal peptide at the N-terminus was retrieved from the C. sinensis transcriptome bank. Recombinant CsAg17 B-cell epitope protein and cDNA vaccines were produced and their immune responses were evaluated in FVB mice. The proportional changes of CD3+/CD4+ and CD3+/CD8+ T cells were detected by flow cytometry, and immune effectors were measured by ELISA.ResultsThe CsAg17 mRNA was transcribed at a higher level in C. sinensis adults than in metacercariae. The CsAg17 protein was distributed in the sperms, oral and ventral suckers, and mesenchymal tissues of C. sinensis adults. In mice challenged with C. sinensis metacercariae, vaccination with CsAg17 protein and cDNA resulted in a reduction to 64% and 69% in worm burden, respectively. Both CsAg17 protein and cDNA vaccines increased the proportion of CD3+/CD4+ and CD3+/CD8+ T cells and stimulated the production of Th1 type cytokines such as interleukin (IL)-2, IL-12, and interferon-γ, while maintaining minimum levels of Th2 cytokines. The levels of IgG specific to CsAg17 protein steeply increased in the two vaccinated groups from 2 weeks after immunization. The liver tissue retained good morphology in the mice vaccinated with CsAg17 protein or cDNA, whereas severe inflammation and large serous cysts were observed in the liver of the unvaccinated mice.ConclusionsVaccination with CsAg17 protein and cDNA reduced the pathological changes in the bile duct and liver, and ameliorated the worm burden via cellular and humoral immune responses. Thus, they may serve as good vaccine candidates against C. sinensis infections.

Designing of precise vaccine construct against visceral leishmaniasis through predicted epitope ensemble: A contemporary approach

Visceral leishmaniasis (VL) caused by Leishmania donovani is a fatal parasitic disease affecting primarily the poor population in endemic countries. Increasing number of deaths as well as resistant to existing drugs necessitates the development of an effective vaccine for successful treatment of VL. The present study employed a combinatorial approach for designing monomer vaccine construct against L. donovani by applying forecasted B- and T- cell epitopes from 4 genome derived antigenic proteins having secretory signal peptides and glycophosphatidylinositol (GPI) anchors with ≤ 1 transmembrane helix. The forecasted population coverage of chosen T cell epitope ensemble (combined HLA class I and II) cover 99.14 % of world-wide human population. The predicted 3D structure of vaccine constructs (VC1/VC2) were modeled using homology modeling approach and docked to innate immune receptors TLR-2 and TLR-4 with respective docking energies −1231.4/−910.3 and −1119.4/−1476 kcal/mol. Overall, the aforementioned designed vaccine constructs were found appropriate for including in self-assembly protein nanoparticles (SAPN) for further study in developing cutting-edge precision vaccine against VL in short duration with cost-effective manner.

Screening of Secreted Proteins of Sporisorium reilianum f. sp. zeae for Cell Death Suppression in Nicotiana benthamiana.

Sporisorium reilianum f. sp. zeae (SRZ) is a biotrophic fungus causing head smut in maize. Maize infection with SRZ leads to very little cell death suggesting the presence of cell-death suppressinpg effectors. Several hundred effector proteins have been predicted based on genome annotation, genome comparison, and bioinformatic analysis. For only very few of these effectors, an involvement in virulence has been shown. In this work, we started to test a considerable subset of these predicted effector proteins for a possible function in suppressing cell death. We generated an expression library of 62 proteins of SRZ under the control of a strong constitutive plant promoter for delivery into plant cells via Agrobacterium tumefaciens-mediated transient transformation. Potential apoplastic effectors with high cysteine content were cloned with signal peptide while potential intracellular effectors were also cloned without signal peptide to ensure proper localization after expression in plant cells. After infiltration of Nicotiana benthamiana leaves, infiltration sites were evaluated for apparent signs of hypersensitive cell death in absence or presence of the elicitin INF1 of Phytophthora infestans. None of the tested candidates was able to induce cell death, and most were unable to suppress INF1-induced cell death. However, the screen revealed one predicted cytoplasmic effector (sr16441) of SRZ that was able to reliably suppress INF1-induced cell death when transiently expressed in N. benthamiana lacking its predicted secretion signal peptide. This way, we discovered a putative function for one new effector of SRZ.

Physiological and Proteomic Changes in the Apoplast Accompany Leaf Senescence in Arabidopsis.

The apoplast, i.e. the cellular compartment external to the plasma membrane, undergoes important changes during senescence. Apoplastic fluid volume increases quite significantly in senescing leaves, thereby diluting its contents. Its pH elevates by about 0.8 units, similar to the apoplast alkalization in response to abiotic stresses. The levels of 159 proteins decrease, whereas 24 proteins increase in relative abundance in the apoplast of senescing leaves. Around half of the apoplastic proteins of non-senescent leaves contain a N-terminal signal peptide for secretion, while all the identified senescence-associated apoplastic proteins contain the signal peptide. Several of the apoplastic proteins that accumulate during senescence also accumulate in stress responses, suggesting that the apoplast may constitute a compartment where developmental and stress-related programs overlap. Other senescence-related apoplastic proteins are involved in cell wall modifications, proteolysis, carbohydrate, ROS and amino acid metabolism, signaling, lipid transport, etc. The most abundant senescence-associated apoplastic proteins, PR2 and PR5 (e.g. pathogenesis related proteins PR2 and PR5) are related to leaf aging rather than to the chloroplast degradation program, as their levels increase only in leaves undergoing developmental senescence, but not in dark-induced senescent leaves. Changes in the apoplastic space may be relevant for signaling and molecular trafficking underlying senescence.

A new serine protease family with elastase activity is produced by Streptomyces bacteria.

We found an elastolytic activity in the culture supernatant of Streptomyces sp. P-3, and the corresponding enzyme (streptomycetes elastase, SEL) was purified to apparent homogeneity from the culture supernatant. The molecular mass of purified SEL was approximately 18 kDa as judged by SDS-PAGE analysis and gel-filtration chromatography. Utilizing information from N-terminal amino acid sequencing of SEL and mass spectrometry of SEL tryptic fragments, we succeeded in cloning the gene-encoding SEL. The cloned SEL gene contains a 726 bp ORF, which encodes a 241 amino acid polypeptide containing a putative signal peptide for secretion (28 amino acid) and pro-sequence (14 amino acid). Although the deduced primary structure of SEL has sequence similarity to proteins in the S1 protease family, the amino acid sequence shares low identity (< 31.5 %) with any known elastase. SEL efficiently hydrolyses synthetic peptides having Ala or Val in the P1 position such as N-succinyl-Ala-Ala-(Pro or Val)-Ala-p-nitroanilide (pNA), whereas reported proteases by streptomycetes having elastolytic activity prefer large residues, such as Phe and Leu. Compared of kcat/Km ratios for Suc-Ala-Ala-Val-Ala-pNA and Suc-Ala-Ala-Pro-Ala-pNA with subtilisin YaB, which has high elastolytic activity, Streptomyces sp. P-3 SEL exhibits 12- and 121-fold higher, respectively. Phylogenetic analyses indicate that the predicted SEL protein, together with predicted proteins in streptomycetes, constitutes a novel group within the S1 serine protease family. These characteristics suggest that SEL-like proteins are new members of the S1 serine protease family, which display elastolytic activity.

Bioinformatics Prediction and Comparison of &lt;i&gt;Artocarpus heterophyllus&lt;/i&gt; BRI1 Family Members

Full-length trantogenic sequencing (PRJNA579273) of jackfruit seedling stems and leaves was performed. On this basis, biological information analysis was performed on Jackfruit ( Artocarpus heterophyllus ) BR INSENSITIVE 1 (BRI1) family members AhBRI1 , AhBRL1 and AhBRL2 to translate genes into proteins. Using online analysis tools, jackfruit, Arabidopsis (Arabidopsis Thaliana), Sichuan mulberry (Morus notabilis) and Poplar (Populus trichocarpa) were used to analyze and obtain different physicochemical properties data and the secondary and tertiary structure of the protein. The bioinformatics prediction and comparison between the BRI1 protein families of different species were obtained. The results showed that the amino acid residues were 1 195, 1 170 and 985, and the theoretical isoelectric points were 6.32, 5.77 and 7.08. The protein was weakly acidic, hydrophilic and stable. There are secretory pathway signal peptides or chloroplast transport peptides with transmembrane structure. The main components of the secondary structure are irregular coil and -helix, and the tertiary structure is spiral tubular structure. This study provides a basis for further functional studies on members of the BRI1 protein family of jackfruit.

LmCBP1, a secreted chitin-binding protein, is required for the pathogenicity of Leptosphaeria maculans on Brassica napus

Leptosphaeria maculans is the causal agent of blackleg disease on Brassica napus. Determining the underlying functions of genes required for pathogenesis is essential for understanding the infection process. A chitin-binding protein (LmCBP1) was discovered as a pathogenicity factor for the infection of B. napus by L. maculans through gene knockout using the CRISPR-Cas9 system. Chitin-binding activity was demonstrated through a chitin-protein binding assay. A secreted signal peptide was detected using a yeast secreted-signal peptide trap assay. An increased expression level during the infection stage was also observed, suggesting that LmCBP1 is a secreted protein. The knockout mutants showed decreased infection on B. napus, with reduced pathogenicity on ten cultivars with/without diverse R genes. The mutants were more sensitive to H2O2 compared to wild type L. maculans isolate JN3. This study provides evidence of the virulence of a novel chitin-binding protein LmCBP1 on B. napus through mutants created via the CRISPR-Cas9 system.

Identification and characterization of α-xylosidase involved in xyloglucan degradation in Aspergillus oryzae

Aspergillus oryzae produces hydrolases involved in xyloglucan degradation and induces the expression of genes encoding xyloglucan oligosaccharide hydrolases in the presence of xyloglucan oligosaccharides. A gene encoding α-xylosidase (termed AxyA), which is induced in the presence of xyloglucan oligosaccharides, is identified and expressed in Pichia pastoris. AxyA is a member of the glycoside hydrolase family 31 (GH31). AxyA hydrolyzes isoprimeverose (α-D-xylopyranosyl-(1→6)-D-glucopyranose) into D-xylose and D-glucose and shows hydrolytic activity with other xyloglucan oligosaccharides such as XXXG (heptasaccharide, Glc4Xyl3) and XLLG (nonasaccharide, Glc4Xyl3Gal2). Isoprimeverose is a preferred AxyA substrate over other xyloglucan oligosaccharides. In the hydrolysis of XXXG, AxyA releases one molecule of D-xylose from one molecule of XXXG to yield GXXG (hexasaccharide, Glc4Xyl2). AxyA does not contain a signal peptide for secretion and remains within the cell. The intracellular localization of AxyA may help determine the order of hydrolases acting on xyloglucan oligosaccharides.

The effector GpRbp-1 of Globodera pallida targets a nuclear HECT E3 ubiquitin ligase to modulate gene expression in the host.

SummaryPlant‐parasitic nematodes secrete effectors that manipulate plant cell morphology and physiology to achieve host invasion and establish permanent feeding sites. Effectors from the highly expanded SPRYSEC (SPRY domain with a signal peptide for secretion) family in potato cyst nematodes have been implicated in activation and suppression of plant immunity, but the mechanisms underlying these activities remain largely unexplored. To study the host mechanisms used by SPRYSEC effectors, we identified plant targets of GpRbp‐1 from the potato cyst nematode Globodera pallida. Here, we show that GpRbp‐1 interacts in yeast and in planta with a functional potato homologue of the Homology to E6‐AP C‐Terminus (HECT)‐type ubiquitin E3 ligase UPL3, which is located in the nucleus. Potato lines lacking StUPL3 are not available, but the Arabidopsis mutant upl3‐5 displaying a reduced UPL3 expression showed a consistently small but not significant decrease in susceptibility to cyst nematodes. We observed a major impact on the root transcriptome by the lower levels of AtUPL3 in the upl3‐5 mutant, but surprisingly only in association with infections by cyst nematodes. To our knowledge, this is the first example that a HECT‐type ubiquitin E3 ligase is targeted by a pathogen effector and that a member of this class of proteins specifically regulates gene expression under biotic stress conditions. Together, our data suggest that GpRbp‐1 targets a specific component of the plant ubiquitination machinery to manipulate the stress response in host cells.

A Novel 'Candidatus Liberibacter asiaticus'-Encoded Sec-Dependent Secretory Protein Suppresses Programmed Cell Death in Nicotiana benthamiana.

‘Candidatus Liberibacter asiaticus’ (CLas) is one of the causal agents of citrus Huanglongbing (HLB), a bacterial disease of citrus trees that greatly reduces fruit yield and quality. CLas strains produce an array of currently uncharacterized Sec-dependent secretory proteins. In this study, the conserved chromosomally encoded protein CLIBASIA_03875 was identified as a novel Sec-dependent secreted protein. We show that CLIBASIA_03875 contains a putative Sec- secretion signal peptide (SP), a 29 amino acid residue located at the N-terminus, with a mature protein (m3875) of 22 amino acids found to localize in multiple subcellular components of the leaf epidermal cells of Nicotiana benthamiana. When overexpressed via a Potato virus X (PVX)-based expression vector in N. benthamiana, m3875 suppressed programmed cell death (PCD) and the H2O2 accumulation triggered by the pro-apoptotic mouse protein BAX and the Phytophthora infestans elicitin INF1. Overexpression also resulted in a phenotype of dwarfing, leaf deformation and mosaics, suggesting that m3875 has roles in plant immune response, growth, and development. Substitution mutagenesis of the charged amino acid (D7, R9, R11, and K22) with alanine within m3875 did not recover the phenotypes for PCD and normal growth. In addition, the transiently overexpressed m3875 regulated the transcriptional levels of N. benthamiana orthologs of CNGCs (cyclic nucleotide-gated channels), BI-1 (Bax-inhibitor 1), and WRKY33 that are involved in plant defense mechanisms. To our knowledge, m3875 is the first PCD suppressor identified from CLas. Studying the function of this protein provides insight as to how CLas attenuates the host immune responses to proliferate and cause Huanglongbing disease in citrus plants.

Identification of secretion domain of Neospora caninum profilin

Profilin (PROF) is a small actin-binding protein presented in apicomplexan protozoa. It was previously reported that Neospora caninum profilin (NcPROF) is secreted into the hemolymph of silkworm larvae regardless of the lack of an identified regular secretion signal peptide. To date, which domain is required for its secretion still remains unknown. To this end, we express a fluorescent protein (mCherry) fused with NcPROF at its N-terminus or C-terminus. Both fusion proteins were expressed and secreted into the culture supernatant from Bm5 cells or hemolymph from silkworm larvae, respectively. To further narrow down the C-terminal minimal domain required for its secretion, we constructed three truncated C-terminal domain constructions, ΔN (aa41–163), ΔN1 (aa50–163), and ΔN2 (aa144–163) respectively. All three fusion proteins were detected in the culture supernatant of Bm5 cells and silkworm hemolymph. Surprisingly, a 20-aa C-terminal α-helix domain facilitates the secretion of mCherry, allowing purification of ΔN2-mCherry from silkworm larval hemolymph by affinity chromatography. Taken together, the secretion domain from NcPROF was identified, indicating that can be utilized for the secretory expression of recombinant proteins in the future.

Expression and secretion of active Moringa oleifera coagulant protein in Bacillus subtilis.

Cationic polypeptide proteins found in the seeds of the tropical plant Moringa oleifera have coagulation efficiencies similar to aluminum and ferric sulfates without their recalcitrant nature. Although these proteins possess great potential to augment or replace traditional coagulants in water treatment, harvesting active protein from seeds is laborious and not cost-effective. Here, we describe an alternative method to express and secrete active M. oleifera coagulant protein (MO) in Bacillus subtilis. A plasmid library containing the MO gene and 173 different types of secretory signal peptides was created and cloned into B. subtilis strain RIK1285. Fourteen of 440 clones screened were capable of secreting MO with yields ranging from 55 to 122mg/L of growth medium. The coagulant activity of the highest MO secreting clone was evaluated when grown on Luria broth, and cell-free medium from the culture was shown to reduce turbidity in a buffered kaolin suspension by approximately 90% compared with controls without the MO gene. The clone was also capable of secreting active MO when grown on a defined synthetic wastewater supplemented with 0.5% tryptone. Cell-free medium from the strain harboring the MO gene demonstrated more than a 2-fold reduction in turbidity compared with controls. Additionally, no significant amount of MO was observed without the addition of the synthetic wastewater, suggesting that it served as a source of nutrients for the effective expression and translocation of MO into the medium.

Denovo designing: a novel signal peptide for tat translocation pathway to transport activin A to the periplasmic space of E. coli.

The twin-arginine translocation (Tat) pathway is one of the bacterial secretory strategies which exports folded proteins across the cytoplasmic membrane. In the present study, we designed a novel Tat-signal peptide for secretion of human activin A used as a recombinant protein model here. In doing so, Haloferax volcanii, Halobacterium salinarum, and Escherichia coli Tat specific signal peptides were aligned by ClustalW program to determine conserved and more frequently used residues. After making the initial signal peptide sequence and doing some mutations, efficiency of this designed signal peptide was evaluated using a set of well-known software programs such as TatP, PRED-TAT, and Phobius. Then the best complex between TatC as an initiator protein in Tat secretory machine and the new designed signal peptide connected to activin A with the lowest binding energy was constructed by HADDOCK server, and ΔΔG value of - 5.5kcal/mol was calculated by FoldX module. After that, efficiency of this novel signal peptide for secretion of human activin A to the periplasmic space of E. coli Rosetta-gami (DE3) strain was experimentally evaluated; to scrutinize the activity of the novel signal peptide, Iranian Bacillus Licheniformis α-Amylase enzyme signal peptide as a Sec pathway signal peptide was used as a positive control. The quantitative analysis of western blotting bands by ImageJ software confirmed the high secretion ability of the new designed signal peptide; translocation of 69% of the produced recombinant activin A to the periplasmic space of E. coli. Circular Dichroism (CD) spectroscopy technique also approved the proper secondary structure of activin A secreted to the periplasmic space. The biological activity of activin A was also confirmed by differentiation of K562 erythroleukemia cells to the red blood cell by measuring the amount of hemoglobin or Fe2+ ion using ICP method. In conclusion, this novel designed signal peptide can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.

In silico analysis of signal peptides for secretory production of a-amylase in Bacillus subtilis

α-Amylases are important commercial enzymes and have a broad application in industrial processes and medicine. Gram-positive bacteria such as Bacillus subtilis are possible host organisms for α-amylases secretory production. Secretion of α-amylases to the culture medium versus intracellular production has several advantages such as prevention of inclusion bodies accumulation, higher product stability and solubility. Signal peptides are considered as one of the most essential elements for successful secretory synthesis of the recombinant proteins. Therefore, by the selection of an efficient signal peptide, secretion of the recombinant protein can be enhanced. The goal of this investigation was the in silico evaluation of several peptides to find the most suitable leader peptides for secretory production of α-amylase in B. subtilis. In present work, 30 signal peptides were selected, and numerous online servers such as SignalP, ProtParam, SOLpro, PRED-TAT and ProtComp was used for investigation of suitable signal peptides. According to in silico predictions all other signal peptides connected to α-amylase were stable and soluble except PPBD_BACSU. PPBD_BACSU because of having D-score below cut-off could not be recognized as a suitable signal peptide for α-amylase. Computational analysis identified QOX2_BACSU may direct protein into transmembrane location and was ignored. All 28 remained were predicted as secretory signal peptides which can excrete protein out of the bacteria. The signal peptides recommended by the present study are valuable for rational designing of secretory soluble α-amylase. Although, such information can be useful for future experimental production of these mentioned secretory proteins.

Cloning and Characterization of a Chondroitin AC Exolyase from Arthrobacter sp. SD-04.

Glycosaminoglycans (GAGs) and their low-molecular weight derivates have received considerable interest in terms of their potential clinical applications, and display a wide variety of pharmacological and pharmacokinetic properties. Structurally distinct GAG chains can be prepared by enzymatic depolymerization. A variety of bacterial chondroitin sulfate (CS) lyases have been identified, and have been widely used as catalysts in this process. Here, we identified a putative chondroitin AC exolyase gene, AschnAC, from an Arthrobacter sp. strain found in a CS manufacturing workshop. We expressed the enzyme, AsChnAC, recombinantly in Escherichia coli, then purified and characterized it in vitro. The enzyme indeed displayed exolytic cleavage activity toward HA and various CSs. Removing the putative N-terminal secretion signal peptide of AsChnAC improved its expression level in E. coli while maintaining chondroitin AC exolyase activity. This novel catalyst exhibited its optimal activity in the absence of added metal ions. AsChnAC has potential applications in preparation of low-molecular weight GAGs, making it an attractive catalyst for further investigation.

Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis.

Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5' untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax increased from 5.11 μM s-1 in the wild type to 7.56 μM s-1 in the mutant R157T.IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.

In silico Analysis of Different Signal Peptides for Secretory Production of Arginine Deiminase in Escherichia coli.

Secretory production of recombinant protein in bacterial hosts fulfills several advantages. Selecting an appropriate secretory signal peptide is a critical step in secretory production of different protein. Several patents report the usage of signal peptides for secretory production of recombinant proteins in E. coli. In silico identification of suitable signal peptides is a reliable and cost-effective alternative to experimental approaches. This study was aimed to predict best signal peptides for the secretory production of recombinant arginine deiminase in E. coli. In this study, 30 different signal peptide sequences were retrieved from database. The signal peptide probability, location of cleavage sites, and n, h and c regions were predicted by SignalP 4.1 and Phobius servers. After purging the 30 predicted secretory signal peptides, TorT, bla, NrfA, TolB, PapC, PldA, Lpp were removed. Several physicochemical properties of the remaining potential SPs were determined by ProtParam, PROSO II, and SOLpro servers for theoretically selecting the best candidates. Based on physicochemical properties, the signal peptides of OmpC, OmpF, and DsbA were identified respectively as the promising candidates for efficient secretory production of arginine deiminase in E. coli. Although the computational approach has established itself as a basis of modern biotechnology, the experimental study is necessary to validate its results. The criteria used in this study could be applied to other targets for recombination processes.

Characterization and expression in Pichia pastoris of a raw starch degrading glucoamylase (GA2) derived from Aspergillus flavus NSH9

The Aspergillus flavus NSH9 gene, encoding a pH and thermostable glucoamylase with a starch binding domain (SBD), was expressed in Pichia pastoris to produce recombinant glucoamylase (rGA2). The full-length glucoamylase gene (2039 bp), and cDNA (1839 bp) encode a 612 amino acid protein most similar to glucoamylase from Aspergillus oryzae RIB40; the first 19 amino acids are presumed to be a signal peptide for secretion, and the SBD is at the C-terminal. The cDNA was successfully secreted by Pichia at 8.23 U mL-1, and the rGA2 was found to be: a 80 kDa monomer, stable from pH 3.0-9.0, with optimum catalytic activity at pH 5.0, active at temperatures up to 80°C (rGA2 retained 58% of its activity after 60 min of incubation at 70°C), and metal ions such as Na+, K+, Ca++ and Mg++ enhanced rGA2 enzyme activity. The starch degrading ability of rGA2 was also observed on raw sago starch and where prolonged incubation generated larger, deeper, holes on the starch granules, indicating rGA2 is an excellent candidate for industrial starch processing applications.

Characterization of the Candida orthopsilosis agglutinin-like sequence (ALS) genes.

Agglutinin like sequence (Als) cell-wall proteins play a key role in adhesion and virulence of Candida species. Compared to the well-characterized Candida albicans ALS genes, little is known about ALS genes in the Candida parapsilosis species complex. Three incomplete ALS genes were identified in the genome sequence for Candida orthopsilosis strain 90–125 (GenBank assembly ASM31587v1): CORT0C04210 (named CoALS4210), CORT0C04220 (CoALS4220) and CORT0B00800 (CoALS800). To complete the gene sequences, new data were derived from strain 90–125 using Illumina (short-read) and Oxford Nanopore (long-read) methods. Long-read sequencing analysis confirmed the presence of 3 ALS genes in C. orthopsilosis 90–125 and resolved the gaps located in repetitive regions of CoALS800 and CoALS4220. In the new genome assembly (GenBank PQBP00000000), the CoALS4210 sequence was slightly longer than in the original assembly. C. orthopsilosis Als proteins encoded features well-known in C. albicans Als proteins such as a secretory signal peptide, N-terminal domain with a peptide-binding cavity, amyloid-forming region, repeated sequences, and a C-terminal site for glycosylphosphatidylinositol anchor addition that, in yeast, suggest localization of the proteins in the cell wall. CoAls4210 and CoAls800 lacked the classic C. albicans Als tandem repeats, instead featuring short, imperfect repeats with consensus motifs such as SSSEPP and GSGN. Quantitative RT-PCR showed differential regulation of CoALS genes by growth stage in six genetically diverse C. orthopsilosis clinical isolates, which also exhibited length variation in the ALS alleles, and strain-specific gene expression patterns. Overall, long-read DNA sequencing methodology was instrumental in generating an accurate assembly of CoALS genes, thus revealing their unconventional features and first insights into their allelic variability within C. orthopsilosis clinical isolates.

Modulation of Cell Adhesion and Differentiation on Collagen Gels by the Addition of the Ovalbumin Secretory Signal Peptide.

Ovalbumin (OVA) is the most abundant protein in egg whites that is unnecessary in the egg yolk-based food industry. The development of OVA-based functional materials is of great interest. Collagen is a major component of the extracellular matrix. In this study, an OVA fragment, the OVA secretory signal peptide (OVA SP), was loaded in collagen gels, which were used as a cell scaffold for various types of cells. We examined the effect of OVA SP loaded in collagen gels to cell properties. The peptide was initially bound to the collagen fibers and then released from the gel. Our results indicate that the released OVA SP suppressed the integrin-mediated cell adhesion and focal adhesion formation. However, the adhesion of NIH3T3 cells was not suppressed by treatment with a chelating agentand an anti-β1 antibody. Our resultssuggest that OVA SP nonspecifically interacts with cell surface proteins. The adhesion of various cell types on collagen gels were changed by the addition of OVA SP, depending on their integrin expression pattern. Additionally, the differentiation of MC3T3-E1 osteoblastic cells was promoted on the OVA SP-loaded collagen gels. This suggests that OVA SP may modulate both the differentiation and the adhesion of cells cultured on the collagen gels.