Prokaryotic Expression System Research Articles

Prokaryotic expression, purification and identification of the N protein of middle east respiratory syndrome coronavirus

Objective To express the N protein of middle east respiratory syndrome coronavirus （ MERS-CoV） in prokaryotic expression system and evaluate the immunogenicity of the purified recombinant N protein.Methods The gene encoding N protein of MERS-CoV was synthesized and cloned into the vector pET30a to construct the recombinant expression plasmid pET30a-MERS-CoV-N.The transformed E.coli BL21 （ DE3） strains carrying expression plasmid were induced by IPTG to express N protein.The expressed protein was purified by using affinity chromatography.SDS-PAGE and Western blot assays were used to iden-tify the expressed N protein and evaluate its immunogenicity.Results The recombinant N protein was suc-cessfully expressed in soluble form with the size of 46×103 .The results of Western blot assay showed that the recombinant N protein could specifically react with rabbit serum samples positive for antibodies against N protein B-cell epitope peptide and mouse anti-His tag antibodies.No positive band against N protein was found when primary antibody was used with thirty adult serum samples from Beijing in 2008.Conclusion N protein of MERS-CoV was successfully expressed in prokaryotic expression system.The successful expres-sion of N protein laid the foundation for immunological diagnosis of N protein of MERS-CoV and researches on its immunogenicity. Key words: Middle east respiratory syndrome coronavirus; N protein; Prokaryotic expression; Purification ; Identification

Identification of Arylphorin interacting with the insecticidal protein PirAB from Xenorhabdus nematophila by yeast two-hybrid system.

PirAB toxin was initially found in the Photorhabdus luminescens TT01 strain and is a demonstrated binary toxin with high insecticidal activity. In this paper, we co-expressed the pirAB gene of Xenorhabdus nematophila HB310 in a prokaryotic expression system, and we found that the PirAB protein showed high hemocoel insecticidal activity against Galleria mellonella, Helicoverpa armigera and Spodoptera exigua. LD50 values were 1.562, 2.003 and 2.17μg/larvae for G. mellonella, H. armigera, and S. exigua, respectively (p > 0.05). Additionally, PirAB-interaction proteins were identified from G. mellonella by 6 × His Protein Pulldown combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of which, arylphorin of G. mellonella showed the highest matching rate. A protein domain conservative structure analysis indicated that arylphorin has three domains including Hemocyanin-N, Hemocyanin-M, and Hemocyanin-C. Among these protein domains, Hemocyanin-C has immune and recognition functions. Further, Hemocyanin-C domain of arylphorin was identified to interact with PirA but not PirB by Yeast two-hybrid system. These findings reveal, for the first time, new host protein interacting with PirAB. The identification of interaction protein may serve as the foundation for further study on the function and insecticidal mechanism of this binary toxin from Xenorhabdus.

Mutagenesis for Improvement of Activity and Stability of Prolyl Aminopeptidase from Aspergillus oryzae.

In this study, the prokaryotic expression system of Escherichia coli was used to modify prolyl aminopeptidase derived from Aspergillus oryzae JN-412 (AoPAP) via random mutagenesis and site-directed saturation mutagenesis. A random mutant library with a capacity of approximately 3000 mutants was compiled using error-prone polymerase chain reaction, and nonconservative amino acids within 3Å of the substrate L-proline-p-nitroaniline were selected as site-directed saturation mutagenesis sites via homologous simulation and molecular docking of AoPAP. Variants featuring high catalytic efficiency were screened by a high-throughput screening method. The specific activities of the variants of 3D9, C185V, and Y393W were 127Umg-1, 156Umg-1, and 120Umg-1, respectively, which were 27%, 56%, and 20% higher than those of the wild type, with a value of 100Umg-1. The half-life of thermostability of the mutant 3D9 was 4.5h longer than that of the wild type at 50°C. The mutant C185V improved thermostability and had a half-life 2h longer than that of the wild type at a pH of 6.5. Prolyl aminopeptidase had improved stability within the acidic range and thermostability after modification, making it more suitable for a synergistic combination with various acidic and neutral endoproteases.

TLR Agonist rHP-NAP as an Adjuvant of Dendritic Cell-Based Vaccine to Enhance Anti-Melanoma Response.

Melanoma is a common and malignant cutaneous tumor, which is responsible for a large proportion of skin cancer deaths. Dendritic cell (DC)-based vaccines have achieved positive results in the treatment of melanoma because of their ability to induce cytotoxic response to facilitate tumor elimination. To improve the efficacy of dendritic cell-based vaccines by the adjuvant activity of Helicobacter pylori neutrophil activating protein (HP-NAP). The recombinant HP-NAP (rHP-NAP) was expressed by using prokaryotic expression system. DCs were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. After treating with rHP-NAP, the maturation of DCs and dendritic cell-based vaccine were assayed by using flow cytometry and qRT-PCR. The activation and proliferation of T cells were measured by FCM, ELISA and MTT methods. The tumor specific cytotoxic response to resistant B16F10 was detected by using lactate dehydrogenase-release assay and qRT-PCR. The rHP-NAP, prepared from E. coli prokaryotic expression system, was able to significantly promote the maturation of dendritic cell-based vaccine loaded with tumor cell lysate (TCL) of B16F10 (DC-B16F10-TCL). Furthermore, it effectively induced the activation and proliferation of T cells and tumor specific cytotoxic response to resistant B16F10 melanoma tumor cells. These results suggested that rHP-NAP possesses the potential for use as an adjuvant of dendritic cell-based vaccine in anti-melanoma treatment.

N-terminal region of Helicobacter pylori CagA induces IL-8 production in gastric epithelial cells via the β1 integrin receptor

Introduction. Helicobacter pylori is associated with gastrointestinal disease, most notably gastric cancer. Cytotoxin-associated antigen A (CagA), an important virulence factor for H. pylori pathogenicity, induces host cells to release inflammatory factors, especially interleukin-8 (IL-8). The mechanism by which C-terminal CagA induces IL-8 production has been studied extensively, but little is known about the role of the N-terminus.Aim. To investigate the effect of CagA303-456aa (a peptide in the N-terminal CagA) on IL-8 production by gastric epithelial cells.Methodology. CagA303-456aa was produced by a prokaryotic expression system and purified by Strep-tag affinity chromatography. An integrin β1 (ITGB1)-deficient AGS cell line was constructed using the CRISPR/Cas9 technique, and NCTC 11637 cagA and/or cagL knockout mutants were constructed via homologous recombination. The levels of IL-8 production were determined by enzyme-linked immunosorbent assay (ELISA), and p38 and ERK1/2 phosphorylation were examined by Western blot.Results. CagA303-456aa induced IL-8 expression by AGS cells. IL-8 induction by CagA303-456aawas specifically inhibited by ITGB1 deficiency. Notably, CagA303-456aa activated the phosphorylation of both p38 and ERK1/2, and blocking p38 and ERK1/2 activity significantly reduced IL-8 induction by CagA303-456aa. ITGB1 deficiency also inhibited the activation of p38 phosphorylation by CagA303-456aa. Finally, experiments in CagA and/or CagL knockout bacterial lines demonstrated that extracellular CagA might induce IL-8 production by AGS cells.Conclusion. Residues 303-456 of the N-terminal region of CagA induce IL-8 production via a CagA303-456-ITGB1-p38-IL-8 pathway, and ERK1/2 is also involved in the release of IL-8. Extracellular CagA might induce IL-8 production before translocation into AGS cells.

Serodiagnosis of Fasciola gigantica Infection in Buffaloes with Native Cathepsin-L Proteases and Recombinant Cathepsin L1-D

Serodiagnosis of Fasciola gigantica natural infection in buffaloes with recombinant cathepsin L1-D and native cathepsin-L protease antigens. The recombinant cat L1-D antigen was expressed in prokaryotic expression system and native cathepsin-L proteases were purified by alcoholic fractionation from adult F. gigantica flukes. Buffaloes (n =325) were screened for anti-Fasciola antibodies with the above antigens in immunoglobulin-G-enzyme linked immunosorbent assay (IgG-ELISA). The recombinant cat L1-D antigen showed positive reactivity with 101/122 necropsy positive animals but 21/122 necropsy confirmed positive animals were negative in this ELISA (sensitivity 82.8%). However, 30/203 (14.8%) necropsy negative animals for Fasciola were seropositive with specificity of 85.2%. With native cat-L protease, 104/122 necropsy confirmed positive animals were ELISA positive but 18/122 necropsy positive animals were seronegative, thereby depicting the sensitivity of 85.2%. But ELISA with this antigen showed 27/203 (13.3%) necropsy negative animals as positive (specificity 86.7%). Comparative evaluation of both the antigens showed that they are suitable for serodiagnosis of F. gigantica infection in buffalo herds.

Expression, purification and functional assessment of asprosin inclusion body

The obtain purified recombinant asprosin and test its functions. The recombinant plasmid of pET-22b-asprosin was constructed and transformed into competent E.coli BL (DE3) strain. After IPTG-induced expression, asprosin inclusion body was renatured by gradient urea and purified by Ni-NTA affinity chromatography column followed by removal of endotoxin to obtain recombinant asprosin for use in cells and animals experiments. C57 mice were injected intraperitoneally with the recombinant asprosin and blood glucose was detected using a blood glucose meter. Alamar Blue assay was used to evaluate of the effect of the recombinant asprosin on the viability of MIHA cells, and cellular glycogen content was detected using the anthrone method. At the absorbance at 600 nm of 0.8, induction of the recombinant host bacteria with 1 mmol/L IPTG at 37 ℃ for 4 h optimally induced the expression of asprosin inclusion body. After purification and endotoxin removal, the purity of the recombinant asprosin exceeded 95% with the content of endotoxin below 1 EU/mg. In C57 mice, intraperitoneal injection with recombinant asprosin significantly increased blood glucose level, which reached the peak level at 60 min following the injection (P=0.021) and recovered the normal level at 120 min (P=0.03). Treatment with the recombinant asprosin for 24 h did not cause obvious adverse effect on the viability of MIHA cells but significantly lowered glycogen content in the cells (P < 0.05). We successfully obtained recombinant asprosin using a prokaryotic expression system. The recombinant asprosin can decrease glycogen content in MIHA cells and increase blood glucose level in mice.

Direct characterization of overproduced proteins by native mass spectrometry

Proteins derived by recombinant technologies must be characterized to ensure quality, consistency and optimal production. These properties are usually assayed following purification procedures that are time consuming and labor intensive. Here, we describe a native mass spectrometry (MS) approach, direct-MS, for rapid characterization of intact overexpressed proteins immediately from crude samples. In this protocol, we discuss the multiple applications of the method and outline the necessary steps required for sample preparation, data collection and interpretation of results. We begin with the sample preparation workflows, which are relevant for recombinant proteins produced within bacteria, those analyzed straight from crude cell lysate, and secreted proteins generated in eukaryotic expression systems that are assessed directly from the growth culture medium. We continue with the mass acquisition steps that enable immediate definition of properties such as expressibility, solubility, assembly state, folding, overall structure, stability, post-translational modifications and associations with biomolecules. We demonstrate the applicability of the method by presenting the characterization of a computationally designed toxin-antitoxin heterodimer, activity and protein-interaction determination of a regulatory protein and detailed glycosylation analysis of a designed intact antibody. Overall, we describe a simple and rapid protocol that is relevant to both prokaryotic and eukaryotic expression systems and can be carried out on multiple mass spectrometers, such as Orbitrap and quadrupole time-of-flight (QTOF)-based mass spectroscopy platforms, that enable intact protein detection. The procedure takes from 30 min to several hours, from sample collection to data acquisition, depending on the depth of MS analysis.

Nimble Cloning: A Simple, Versatile, and Efficient System for Standardized Molecular Cloning.

Molecular cloning is one of the most fundamental technologies in molecular biology, and has been critical for driving biotechnological advances. In this study, we have developed a novel method for standardized molecular cloning. The cloning technique known as “Nimble Cloning” uses the restriction enzyme, SfiI, in combination with the T5 exonuclease, to linearize the vector and generate 3′-overhangs simultaneously. Both PCR products and plasmids can be used for the cloning reaction in the Nimble Cloning system. The cloning system is highly efficient, suitable for gene expression in both prokaryotic and eukaryotic expression systems, and enables the reuse of DNA fragments or plasmid entry clones. Nimble Cloning is applicable for the cloning of single or multiple fragments, as well as multi-site cloning. Due also to its simplicity and versatility, the cloning method has great potential for the modular assembly of DNA constructs.

Construction and immune efficacy of recombinant Lactobacillus casei strains expressing Malt from Aeromonas veronii

Aeromonas veronii is an important zoonotic pathogen that causes significant economic losses in the aquaculture industry. The use of probiotics in aquaculture is a practical alternative to antibiotics to promote animal health and aid in disease prevention. In the present study, we aimed to construct a recombinant Lactobacillus casei(surface-displayed or secretory) strain containing Malt from A. veronii TH0426 and assess its potential as an oral vaccine. A 1314-bp Malt gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in resulting recombinant strains Lc-MCS-Malt (surface-displayed) and Lc-pPG-Malt (secretory) was then verified by Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, with the molecular weight of the corresponding protein shown to be 48 kDa. Furthermore, a fluorescent signal for Lc-MCS-Malt was observed by fluorescence microscopy. At 0, 7, 16, 25, and 34 days post-immunization, tissue and blood samples were collected from common carp orally administered with the recombinant L. casei strains for immune-related index analyses. Treatment of common carp with the recombinant vaccine candidate stimulated high serum or skin mucus specific antibody titers and induced a higher lysozyme, ACP, SOD activity, while fish fed with Lc-pPG or PBS had no detectable immobilizing immune responses. Expression of IL-10, IL-1β, TNF-α, and IFN-γ genes in the group immunized with recombinant L. casei were significantly (P < 0.05) up regulated as compared with control groups, indicating that inflammatory response and cell immune response were triggered. Results also showed that recombinant L. casei could stimulate the mucosa through colonization of the intestine, resulting in increased transcription of IL-10, IL-1β, TNF-α, and IFN-γ. Immunity and colonization assays also showed that after 34 days of fasting, recombinant L. casei were still present in the intestines of the immunized fish. Common carp that received Lc-MCS-Malt(53.3%) and Lc-pPG-Malt (46.7%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our findings suggested that recombinant L. casei can adequately protect fish and improve immunity, providing a theoretical basis for the future development of an oral Lactobacillus vaccine for use in aquaculture.

Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System.

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

Active immunization against AMH reveals its inhibitory role in the development of pre-ovulatory follicles in Zhedong White geese

The aim of this study was to investigate the effects of active immunization against recombinant Anti-Müllerian hormone (AMH) protein on the ovarian follicular development, egg production, and molecular regulatory mechanisms in broody-prone Zhedong White geese. For this, a recombinant goose AMH protein was expressed using a prokaryotic expression system. Fifty incubating geese from the same genetic background were selected and equally divided into two groups. The immunization group was actively immunized against the recombinant goose AMH protein, whereas the control group was immunized against bovine serum albumin (BSA). Immunization against AMH accelerated ovarian follicular development and increased clutch sizes by one to two eggs in two consecutive laying-incubation cycles. Furthermore, immunization against AMH upregulated the mRNA transcription levels of the FSH-beta gene in the pituitary gland, and FSHR, 3beta-HSD, and Smad4 genes in the granulosa layer of pre-ovulatory follicles; however, immunization downregulated the expression of the OCLN gene in the granulosa layer of pre-ovulatory follicles, and Smad5 and Smad9 genes in the granulosa layer of SYFs. These results suggest that AMH might hinder ovarian follicular development by decreasing both pituitary FSH secretion as well as ovarian follicular sensitivity to FSH. The latter molecular mechanism could be fulfilled by regulating Smad5 or Smad9 signals in SYFs, as well as the FSHR and Smad4 signals that affect progesterone synthesis and yolk deposition in the pre-ovulatory follicles.

Synergistic activation of Src, ERK and STAT pathways in PBMCs for Staphylococcal enterotoxin A induced production of cytokines and chemokines.

Staphylococcal enterotoxin A (SEA) is a well-known superantigen and stimulates human peripheral blood mononuclear cells (PBMCs) involving in the pathogenesis of inflammatory disorders and cancer. To better understand the biological activities of SEA and the possible intracellular mechanisms by which SEA plays its roles in conditions like staphylococcal inflammatory and/or autoimmune disorders and immunotherapy. Recombinant SEA (rSEA) was expressed in a prokaryotic expression system and its effects on the cytokine and chemokine production was examined by Enzyme-linked Immunospot (ELISpot) Assay and ELISA analysis. In vitro experiments showed rSEA could significantly enhance secretion of a broad spectrum of cytokines and chemokines from PBMCs dose-dependently. Increased secretion of cytokines and chemokines from rSEA stimulated PBMCs was barely affected by C-C motif chemokine receptor 2 (CCR2) antagonist INCB3344. However, Src, ERK and STAT pathway inhibitors were able to successfully block the enhanced secretion of most of cytokines and chemokines produced by rSEA stimulated PBMCs. Our work suggested that rSEA serves as a potent stimulant of PBMCs, and induces the release of cytokines and chemokines through Src, ERK and STAT pathways upon a relatively independent network. Our work also strongly supported that Src, ERK and STAT signaling inhibitors could be effective therapeutic agents against diseases like toxic shock syndrome or infection by microbes resistant to antibiotics.

Recombinant Allergens – Production Methods and Use in Molecular Diagnosis and Allergen Immunotherapy

Although allergen extracts show some disadvantages, they are widely used both for in vivo diagnosis and for allergen immunotherapy. One way to overcome allergen extracts’ limitations is to use recombinant allergens. Selection of an appropriate expression system is dependent on the characteristics and intended application of the recombinant protein. The prokaryotic expression system is suitable for allergens that do not require post-translational modifications; however, there are allergens that need to be expressed in eukaryotic cells in order to acquire their biological activity. The use of these recombinant proteins ensures an increased specificity and sensitivity of the diagnosis and safer and more effective immunotherapy.

Evaluation of recombinant human vascular endothelial growth factor VEGF121-loaded poly-l-lactide microparticles as a controlled release delivery system.

Vascular endothelial growth factor A (VEGF-A) is an important growth factor that plays a major role in angiogenesis. With different isoforms distributed in various tissues, the shortest isoform of VEGF-A is VEGF121, one of the physiologically functional variants next to VEGF165. It is well known that VEGF has a shorter half-life, and the stability of the protein must be considered in therapeutic aspects. Poly-l-lactide (PLA) microparticles can release the encapsulated protein in a sustained release mode. In this study, the VEGF121 gene was cloned and expressed in a prokaryotic expression system (Escherichia coli). The recombinant VEGF121 was encapsulated with PLA microparticles and studied in vitro and ex ovo for the sustained release mechanism. The PLA-VEGF microparticles and the recombinant VEGF121 were explored for their bioactivity in human umbilical vein endothelial cells (HUVEC). VEGF released in vitro from PLA microparticles on days 1, 20, and 30 showed remarkable biological activity compared to PBS-loaded PLA microparticles such as the ability of the cells to proliferate, migrate, and form tubes similar to recombinant VEGF121. Besides, PLA-VEGF microparticles and the recombinant VEGF121 were also tested for their proangiogenic action in embryonated eggs by chicken chorioallantoic membrane assay (CAM), and the effect was observed in both forms. This study suggests that PLA-loaded VEGF microparticles in a sustainable release format can be effectively used in proangiogenic therapy and reduce the adverse effects caused due to multiple dosages.

Yeast surface displayed xylanase is an efficient strategy to induce corn defence response

As a valuable and powerful platform technology, yeast surface display (YSD) has been widely used in various biotechnological fields, but little in agricultural applications. Xylanase can be used as an elicitor in the plant–pathogen interaction system and induce corn defence response. In this study, the YSD technology was used to express the xylanase protein family from Setosphaeria turcica, which is a novel strategy for plant protection. First, five genes encoding xylanase were selected from the S. turcica genome database. The conserved domains of these genes were analyzed, which revealed that these genes were highly conserved. Here, we selected two of the five genes and expressed them by a prokaryotic expression system, but the two xylanase genes were expressed in inclusion bodies and showed no activity. To make them available in the application, we successfully constructed them in a YSD system. The products were detected for enzyme activities and the activities of two xylanases were 16.72 and 19.12 U/mL, respectively. We finally proved that the yeast-displaying xylanase was an efficient strategy to induce corn defence response, as four defence genes in corn, PR-1, PR-4, SOD and CAT were induced after treatment with the yeast displaying xylanases.

In silico Analysis, Molecular Docking, Molecular Dynamic, Cloning, Expression and Purification of Chimeric Protein in Colorectal Cancer Treatment.

IntroductionColorectal cancer (CRC) is a type of cancer in humans that leads to high mortality and morbidity. CD166 and CD326 are immunoglobulins that are associated with cell migration. These molecules are included in tumorigenesis of CRC and serve a great marker of CRC stem cells. In the present study, we devised a novel chimeric protein including the V1-domain of the CD166 and two epitopes of CD326 to use in diagnostic or therapeutic applications.MethodsIn silico techniques were launched to characterize the properties and structure of the protein. We have predicted physicochemical properties, structures, stability, MHC class I binding properties and ligand-receptor interaction of this chimeric protein by means of computational bioinformatics tools and servers. The sequence of chimeric gene was optimized for expression in prokaryotic host using online tools and cloned into pET-28a plasmid. The recombinant pET28a was transformed into the E. coli BL21DE3. Expression of recombinant protein was examined by SDS-PAGE and Western blotting.ResultsThe designed chimeric protein retained high stability and the same immunogenicity as of the original proteins. Bioinformatics data indicated that the epitopes of the synthetic chimeric protein might induce B-cell- and T-cell-mediated immune responses. Furthermore, a gene was synthesized using the codon bias of a prokaryotic expression system. This synthetic gene expressed a bacterial expression system. The recombinant protein with molecular weights of 27kDa was expressed and confirmed by anti-his Western blot analysis.ConclusionThe designed recombinant protein may be useful as a CRC diagnostic tool and for developing a protective vaccine against CRC.

Septin14, a gene specifically expressed in the testis and seminal vesicle of the Banna mini-pig inbred line (BMI).

Septin14 is an important spermatogenesis related gene involved in the pathogenesis of male infertility that has not been well studied. Here, full-length Septin14 cDNA of the Banna mini-pig inbred line (BMI) was cloned using the RACE method and expressed in pig kidney epithelial cells (PK15) and E. coli Rosetta (DE3) cells. Septin14 expression was identified in somatic tissues and testis in different developmental stages. The pig Septin14 CDS is 1,299 bp long, and encodes a peptide (or protein) of 432 amino acids (MW=50.4 kDa). Phylogenetic analysis indicated that pig Septin14 was highly evolutionarily conserved. Subcellular localization of GFP-tagged Septin14 fusion protein revealed that Septin14 was distributed throughout the testicular cells. Among 34 pig tissues, Septin14 mRNA was found specifically in testis and seminal vesicle. In six different postnatal developmental stages, the testicular level of Septin14 mRNA was barely detectable on day 2, while the highest level occurred on day 75. The spatiotemporal expression profile of Septin14, reported herein for the first time in pig, indicated that Septin14 might be involved in the division, development and apoptosis of germ cells. Furthermore, using a pET prokaryotic expression system, we expressed and isolated recombinant 67.9 kDa Septin14 protein.

Cloning, Expression and Purification of &lt;i&gt;MGG_14095&lt;/i&gt; Gene of &lt;i&gt;Magnaporthe grisea&lt;/i&gt;

Magnaporthe grisea is the most harmful pathogen in the world for rice and the main model organism for elucidating the molecular basis of plant fungal disease interaction. Carrying out relevant research of the growth and development of Magnaporthe grisea has a great significance for the control of Magnaporthe grisea . MGG _ 14095 gene is the pathogenic gene of Magnaporthe grisea . The bioinformatics prediction of MGG_14095 protein showed that the isoelectric point (PI) of MGG_14095 protein was 5.72, and the instability index was 50.99, belonging to the acid unstable protein and the α/β hydrolase superfamily, containing the keratinase conservative domain; having obvious transmembrane structure and signal peptide cutting site, belonging to the secretory protein. In this study, the MGG _ 14095 ( 38 ~ 281 ) gene of Magnaporthe grisea was cloned, and the prokaryotic expression system pETM20- MGG _ 14095 ( 38 ~ 28 ) was constructed. We use the stepwise chromatography to purify MGG_14095 protein, and the high-purity soluble target protein was obtained successfully. The results provided for the analysis of MGG_14095(38~281) protein structure, the exploration of pathogenetic mechanism of Magnaporthe grisea and the control of Magnaporthe grisea Certain theoretical and experimental basis.

Identification and Characterization of an OSH1 Thiol Reductase from Populus Trichocarpa.

Interferon gamma-induced lysosomal thiol reductase (GILT) is abundantly expressed in antigen-presenting cells and participates in the treatment and presentation of antigens by major histocompatibility complex II. Also, GILT catalyzes the reduction of disulfide bonds, which plays an important role in cellular immunity. (1) Background: At present, the studies of GILT have mainly focused on animals. In plants, GILT homologous gene (Arabidopsis thaliana OSH1: AtOSH1) was discovered in the forward screen of mutants with compromised responses to sulphur nutrition. However, the complete properties and functions of poplar OSH1 are unclear. In addition, CdCl2 stress is swiftly engulfing the limited land resources on which humans depend, restricting agricultural production. (2) Methods: A prokaryotic expression system was used to produce recombinant PtOSH1 protein, and Western blotting was performed to identify its activity. In addition, a simplified version of the floral-dip method was used to transform A. thaliana. (3) Results: Here, we describe the identification and characterization of OSH1 from Populus trichocarpa. The deduced PtOSH1 sequence contained CQHGX2ECX2NX4C and CXXC motifs. The transcript level of PtOSH1 was increased by cadmium (Cd) treatment. In addition, recombinant PtOSH1 reduced disulfide bonds. A stress assay showed that PtOSH1-overexpressing (OE) A. thaliana lines had greater resistance to Cd than wild-type (WT) plants. Also, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in PtOSH1-OE plants were significantly higher than those in WT A. thaliana. These results indicate that PtOSH1 likely plays an important role in the response to Cd by regulating the reactive oxygen species (ROS)-scavenging system. (4) Conclusions: PtOSH1 catalyzes the reduction of disulfide bonds and behaves as a sulfhydryl reductase under acidic conditions. The overexpression of PtOSH1 in A. thaliana promoted root development, fresh weight, and dry weight; upregulated the expression levels of ROS scavenging-related genes; and improved the activity of antioxidant enzymes, enhancing plant tolerance to cadmium (Cd) stress. This study aimed to provide guidance that will facilitate future studies of the function of PtOSH1 in the response of plants to Cd stress.