His-tagged Fusion Protein Research Articles

Cloning, expression and characterization of C. crescentus xynA2 gene and application of Xylanase II in the deconstruction of plant biomass.

Biotechnology offers innovative alternatives for industrial bioprocesses mainly because it uses enzymes that biodegrade the hemicellulose releasing fermentable sugars. Caulobacter crescentus (C. crescentus) has seven genes responsible for xylanolytic cleavage, 5 to β-xylosidases (EC 3.2.1.37) and 2 for endoxylanases, like xynA2 (CCNA_03137) that encodes Xylanase II (EC 3.2.1.8) of the glycohydrolases-GH10 group. The xynA2 gene was amplified by PCR, cloned into the pTrcHisA vector e efficiently overexpressed in E. coli providing a His-tag fusion protein. Recombinant xylanase (XynA2) was purified by affinity chromatography using a nickel sepharose column and exhibited a single 43kDa band on SDS-PAGE gel. XynA2 showed an optimum alkaline pH (8) and stability at alkaline pH for 24h. Although C. crescentus is mesophilic, XynA2 has optimum temperature of 60°C and is thermo-resistance at 65°C. XynA maintains 66% of the enzymatic activity at high temperatures (90°C) without being denatured.The enzyme displayed a xylanolitic activity free of cellulase to xylan from beechwood and it was not inhibited in the presence of 50μmolmL-1 of xylose. In addition, dithiothreitol (DTT) induced XynA2 activity, as it improved its kinetic parameters by lowering the KM (5.78μmolmL-1) and increasing the KCat/KM ratio (1.63 U s-1). Finally, C. crescentus XynA2 efficiently hydrolyzed corn straw with high release of reducing sugars that can be applied in different branches of the industry.

Rapid detection of 21 β-lactams using an immunochromatographic assay based on the mutant BlaR-CTD protein from Bacillus Licheniformis.

In this study, a gold immunochromatographic assay (GICA) based on a penicillin receptor protein (PBP) is proposed to simultaneously detect penicillin, cephalosporin, and carbapenem antibiotics in milk and chicken. A mutant PBP, carboxy-terminal of the β-lactam sensor-transducer protein (BlaR-CTD, I188KS19CG24C) from Bacillus licheniformis ATCC14580 was expressed in E. coli as a soluble His-tag fusion protein. The purified BlaR-CTD mutant was labeled with colloidal gold (CG-BlaR-CTD-M) and applied to develop GICA. The GICA method was performed based on a competitive form, where the binding of the ampicillin-bovine serum albumin conjugate (Amp-BSA) to CG-BlaR-CTD-M was inhibited by free β-lactams in samples. The performance of the assay was confirmed by analysis of milk and chicken samples. The limits of detection of 21 β-lactam antibiotics met the maximum residue limits set by the European Union and China. The GICA gave results within 10 min, thus providing a rapid and reliable method for the on-site detection of multiple β-lactams in milk and chicken samples.

Expression, purification, characterization and in silico analysis of newly isolated hydrocarbon degrading bleomycin resistance dioxygenase.

In the present investigation, we report cloning, expression, purification and characterization of a novel Bleomycin Resistance Dioxygenase (BRPD). His-tagged fusion protein was purified to homogeneity using Ni-NTA affinity chromatography, yielding 1.2mg of BRPD with specific activity of 6.25 Umg-1 from 600ml of E. coli culture. Purified enzyme was a dimer with molecular weight ~26kDa in SDS-PAGE and ~73kDa in native PAGE analysis. The protein catalyzed breakdown of hydrocarbon substrates, including catechol and hydroquinone, in the presence of metal ions, as characterized via spectrophotometric analysis of the enzymatic reactions. Bleomycin binding was proven using the EMSA gel retardation assay, and the putative bleomycin binding site was further determined by in silico analysis. Molecular dynamic simulations revealed that BRPD attains octahedral configuration in the presence of Fe2+ ion, forming six co-ordinate complexes to degrade hydroquinone-like molecules. In contrary, in the presence of Zn2+ ion BRPD adopts tetrahedral configuration, which enables degradation of catechol-like molecules.

Analysis of lysyl oxidase as a marker for diagnosis of canine mammary tumors.

Lysyl oxidase (LOX) is an extracellular metalloenzyme which mediates crosslinking of collagen and elastin. It has been reported to play a pivotal role in cancer metastasis especially in women suffering from breast cancer. The present study is the first to evaluate the gene expression levels of LOX by Real time-polymerase chain reaction (Real time-PCR) in dogs with mammary tumor besides molecular cloning and expression of canine lysyl oxidase gene (lox). Real time-PCR studies showed a significant upregulation (threefold higher) of lox in mammary tumor cases as compared to healthy dogs indicating its possible diagnostic and prognostic role in canine mammary tumors (CMTs). Cloning and sequencing of lox gene revealed 1230bp CDS which is mostly conserved in C-terminal region. Sequence analysis of canine lox showed that it shares 99% homology with the predicted sequence available on NCBI and had greatest identity with the lox gene from cat. Protein structure predicted with homology modelling was validated by Ramachandran plot analysis which revealed most (approximately 95%) of the amino acids in favoured region. Additionally, recombinant lysyl oxidase expressed as His-tagged fusion protein in prokaryotic expression vector (pPROExHTa) was used in an ELISA for detection of circulating protein LOX in serum of CMT subjects. Receiver operating characteristics analysis of the ELISA revealed high sensitivity (90%) and specificity (85%) with histopathology as reference standard. Taken together, we propose LOX as a diagnostic biomarker and a putative prognostic candidate in CMT cases.

A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study.

Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The kcat/Km value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.

Expression and purification of an ArsM-elastin-like polypeptide fusion and its enzymatic properties

Enzymes could act as a useful tool for environmental bioremediation. Arsenic (As) biomethylation, which can convert highly toxic arsenite [As(III)] into low-toxic volatile trimethylarsine, is considered to be an effective strategy for As removal from contaminated environments. As(III) S-adenosylmethyltransferase (ArsM) is a key enzyme for As methylation; its properties and preparation are crucial for its wide application. Currently, ArsM is usually purified as a His-tag fusion protein restricting widespread use due to high costs. In this study, to greatly reduce the cost and simplify the ArsM preparation process, an Elastin-like polypeptide (ELP) tag was introduced to construct an engineered Escherichia coli (ArsM-ELP). Consequently, a cost-effective and simple non-chromatographic purification approach could be used for ArsM purification. The enzymatic properties of ArsM-ELP were systematically investigated. The results showed that the As methylation rate of purified ArsM-ELP (> 35.49%) was higher than that of E. coli (ArsM-ELP) (> 10.39%) when exposed to 25μmol/L and 100μmol/L As(III), respectively. The purified ArsM-ELP was obtained after three round inverse transition cycling treatment in 2.0mol/L NaCl at 32°C for 10min with the yield reaching more than 9.6% of the total protein. The optimal reaction temperature, pH, and time of ArsM-ELP were 30°C, 7.5 and 30min, respectively. The enzyme activity was maintained at over 50% at 45°C for 12h. The enzyme specific activity was 438.8 ± 2.1U/μmol. ArsM-ELP had high selectivity for As(III). 2-Mercaptoethanol could promote enzyme activity, whereas SDS, EDTA, Fe2+, and Cu2+ inhibited enzyme activity, and Mg2+, Zn2+, Ca2+, and K+ had no significant effects on it.

Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum.

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.

Recombinant expression, purification and bioactivity characterization of extracellular domain of human tumor necrosis factor receptor 1

The interaction between TNF-α with TNFR1 triggers important signaling pathways inducing diverse cellular phenomena including inflammation, apoptosis, etc., and is involved in the pathogenesis and progression of numerous autoimmune diseases. The extracellular domain (ECD) of TNFR has been successfully used to clinically treat such TNF-associated diseases. However, large-scale production of these biological material via eukaryotic cell expression systems is usually costly owing to the culture medium and complicated growth conditions. This study aimed to extract pure soluble human TNFR1-ECD and investigate its biological activity, using a prokaryotic expression system. Recombinant vector pMCSG7-TNFR1-ECD was constructed via ligation-independent cloning. The His-tag fusion protein was expressed in E. coli and localized in inclusion bodies. Recombinant TNFR1-ECD was refolded and purified via nickel-affinity chromatography, tag cleavage, followed by cation-exchange chromatography or size-exclusion chromatography. A purity of over 95% and a yield of 9.3 mg protein per liter of bacterial culture media was obtained. The purified protein showed significant affinity of 2.15 nM towards human TNF-α and inhibited TNF-α-mediated cytotoxicity in L929 cells, with an ED50 of 0.10 μg/ml. It formed a self-associated oligomer with a KD of 1.15 μM, detected via microscale thermophoresis. We thus established a highly efficient approach to construct, express, and purify the recombinant protein of human TNFR1-ECD from a prokaryotic system. The antagonistic bioactivities in vitro indicate this protein as a prospective molecules for drug research against autoimmune diseases characterized by TNF-α overexpression.

Biochemical Characterization of the Rice Cinnamyl Alcohol Dehydrogenase Gene Family.

Cinnamyl alcohol dehydrogenase (CAD) is involved in the final step of the phenylpropanod pathway, catalyzing the NADPH-dependent reduction of hydroxy-cinnamaldehydes into the corresponding alcohols. The rice genome contains twelve CAD and CAD-like genes, collectively called OsCADs. To elucidate the biochemical function of the OsCADs, OsCAD1, 2, 6, and 7, which are highly expressed in rice, were cloned from rice tissues. The cloned OsCADs were heterologously expressed in Escherichia coli as His-tag fusion proteins. The activity assay of the recombinant OsCADs showed that OsCAD2, 6, and 7 have CAD activity toward hydroxycinnamaldehydes, but OsCAD1 has no detectable catalytic activity. The kinetic parameters of the enzyme reactions demonstrated that OsCAD2 has the highest catalytic activity among the examined enzymes. This result agrees well with the finding that the Zn binding and NADPH binding motifs and the residues constituting the substrate binding pocket in bona fide plant CADs were fully conserved in OsCAD2. Although they have large variations in the residue for the substrate binding pocket, OsCAD6 and 7 catalyzed the reduction of hydroxycinnamaldehydes with a similar efficiency. Alignment of amino acid sequences showed that OsCAD1 lacks the GxxxxP motif for NADPH binding and has mismatches in residues important in the reduction process, which could be responsible for the loss of catalytic activity. OsCAD2 belongs to CAD Class I with bona fide CADs from other plant species and is constitutively expressed throughout the developmental stages of rice, with preferential expression in actively lignifying tissues such as the root, stem, and panicle, suggesting that it is mainly involved in developmental lignification in rice. The expression of OsCAD2 was also induced by biotic and abiotic stresses such as Xanthomonas oryzae pv. oryzae (Xoo) infection and UV-irradiation, suggesting that it plays a role in the defense response of rice, in addition to a bona fide role in developmental lignification. OsCAD6 and 7 belong in CAD Class II. Their expression is relatively lower than that of OsCAD2 and is confined to certain tissues, such as the leaf sheath, stem, and panicle. The expression of OsCAD6 was stimulated by Xoo infection and UV-irradiation. Thus OsCAD6 appears to be an inducible OsCAD that is likely involved in the defense response of rice against biotic and abiotic stresses.

Expression, purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243–368)

Zinc finger protein ZNF191(243–368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243–368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243–368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His6-tag system could be used to express ZNF191(243–368). The presence of the His6-tag at the N-terminus of ZNF191(243–368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His6-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.

High-Level Production of High-Purity Human and Murine Recombinant Prion Proteins Functionally Compatible to In Vitro Seeding Assay.

Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with ＞95% purity. The purified recPrPs predominantly adopted an α-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.

A novel solvent tolerant esterase of GDSGG motif subfamily from solar saltern through metagenomic approach: Recombinant expression and characterization

A novel esterase, designated as EstSP was identified by function based screening from a soil metagenomic fosmid library of solar saltern of Goa. EstSP gene of 1065 bp encoding a putative esterase of 354 amino acids showing 55% identity to esterase from gamma proteobacterium HIMB55 was identified. The enzyme EstSP belongs to family IV hormone sensitive lipase with novel sequence characteristics and a unique motif GDSGG. EstSP expressed as a His-tag fusion protein of mass 58 kDa was visualized on SDS PAGE and confirmed by Western blot analysis. The enzyme is an alkaline esterase that exhibited highest catalytic activity towards p-nitrophenyl acetate with optimum temperature 40 °C and pH 8.0. The catalytic efficiency and specific activity of EstSP for p-nitrophenyl acetate was 7407.4 min−1 mM−1 and 915.23 U mg−1 respectively. EstSP showed remarkable stability in the presence of polar and non-polar solvents, retaining >80% of its activity after 72 h. Furthermore, the enzyme is halotolerant with optimum activity at 1 M NaCl and maintained 60% residual activity after 24 h exposure to 5 M NaCl. This novel enzyme with remarkable properties could be a promising candidate for industrial bioprocesses in non-aqueous media as well as pharmaceutical, food and biotechnological applications.

Stability improvement of human collagen α1(I) chain using insulin as a fusion partner

To enhance the stability of recombinant human collagen α1(I) chains (rhCOL1A1) in production and purification stages, a gene fragment fusing COL1A1 and insulin protein coding domains was synthesized and inserted into the pPIC9K expression vector. The fusion peptide-expressing Pichia pastoris strain was created by transformation. After optimization of shake flask cultures, the ultimate intracellular expression level of the insulin-collagen α1(I) chain fusion protein (INS-COL1A1) reached about 300 mg·L− 1, and no obvious protein degradation was found in the fermentation and purification processes. The His-tagged recombinant fusion protein was detected by western blotting and was effectively purified using Ni2 +-chelating chromatography. A prominent improvement in the stability of INS-COL1A1 was observed compared to rhCOL1A1 in vitro, and the rhCOL1A1 released from the fusion protein was studied by LC–MS/MS and in bioassays. The results showed that the purified rhCOL1A1 was consistent with the native protein in amino acid composition and had a similar biological compatibility. To our knowledge, this is the first study to demonstrate the use of insulin as a fusion protein to improve the stability of easily degradable proteins.

Enterotoxigenic Escherichia coli Adhesin-Toxoid Multiepitope Fusion Antigen CFA/I/II/IV-3xSTaN12S-mnLTG192G/L211A-Derived Antibodies Inhibit Adherence of Seven Adhesins, Neutralize Enterotoxicity of LT and STa Toxins, and Protect Piglets against Diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Vaccines inducing antibodies to broadly inhibit bacterial adherence and to neutralize toxin enterotoxicity are expected to be effective against ETEC-associated diarrhea. 6×His-tagged adhesin-toxoid fusion proteins were shown to induce neutralizing antibodies to several adhesins and LT and STa toxins (X. Ruan, D. A. Sack, W. Zhang, PLoS One 10:e0121623, 2015, https://doi.org/10.1371/journal.pone.0121623). However, antibodies derived from His-tagged CFA/I/II/IV-2xSTaA14Q-dmLT or CFA/I/II/IV-2xSTaN12S-dmLT protein were less effective in neutralizing STa enterotoxicity and were not evaluated in vivo for efficacy against ETEC diarrhea. Additionally, His-tagged proteins are considered less desirable for human vaccines. In this study, we produced a tagless adhesin-toxoid MEFA (multiepitope fusion antigen) protein, enhanced anti-STa immunogenicity by including a third copy of STa toxoid STaN12S, and examined antigen immunogenicity in a murine model. Moreover, we immunized pregnant pigs with the tagless adhesin-toxoid MEFA protein and evaluated passive antibody protection against STa+ or LT+ ETEC infection in a pig challenge model. Results showed that tagless adhesin-toxoid MEFA CFA/I/II/IV-3xSTaN12S-mnLTR192G/L211A induced broad antiadhesin and antitoxin antibody responses in the intraperitoneally immunized mice and the intramuscularly immunized pigs. Mouse and pig serum antibodies significantly inhibited adherence of seven colonization factor antigen (CFA) adhesins (CFA/I and CS1 to CS6) and effectively neutralized both toxins. More importantly, suckling piglets born to the immunized mothers acquired antibodies and were protected against STa+ ETEC and LT+ ETEC diarrhea. These results indicated that tagless CFA/I/II/IV-3xSTaN12S-mnLTR192G/L211A induced broadly protective antiadhesin and antitoxin antibodies and demonstrate that this adhesin-toxoid MEFA is a potential antigen for developing broadly protective ETEC vaccines.

Direct interaction between VRN1 protein and the promoter region of the wheat FT gene.

The wheat florigen gene Wheat FLOWERING LOCUS T (WFT, which is identical to VRN3) is an integrator of the vernalization, photoperiod and autonomous pathways in wheat flowering. Many studies have indicated that VERNALIZATION 1 (VRN1) directly or indirectly up-regulates WFT expression in leaves. VRN1 encodes an APETALA1/FRUITFULL-like MADS box transcription factor that is up-regulated by vernalization and aging, leading to promotion of flowering. In this study, the VRN1 protein was expressed as a His-Tag fusion protein in Escherichia coli and used in an electrophoretic mobility shift assay (EMSA). The results from the EMSA indicated that the VRN1 protein directly binds to the CArG-box in the promoter region of WFT, suggesting the direct up-regulation of WFT by VRN1 in the leaves of wheat plants.

Measurement of binding strength between prey proteins interacting with Toxoplasma gondii SAG1 and SAG2 using isothermal titration calorimetry (ITC).

Following the outcome from a previously performed yeast two-hybrid experiment, the binding strength between T. gondii SAG1 and SAG2 and their respective prey proteins were further confirmed in this study. The sag1, sag2 and their prey genes were amplified and cloned into a pGEMT vector. To express the recombinant proteins, the fragments were then subcloned into a pRSETA vector and transformed into E. coli BL21 (DE3) cells. The recombinant proteins were expressed optimally at 37°C and 1mM of IPTG. The 6X His-tag fusion proteins were purified, dialyzed and concentrated. To confirm the expressed proteins, the recombinant proteins were analysed by SDS-PAGE and Western blot. As expected, the size of SAG1, SAG2, HLY and HZF protein were 32, 23, 28 and 37 kDa, respectively. The purified proteins were loaded onto a MicroCal Auto-iTC200 calorimeter from MicroCal™ to quantify binding strength. ITC results indicated there was a typical binding curve for interactions between SAG1 and HLY protein. However, there was an atypical binding curve obtained for interactions between SAG2 and HZF protein. By observing the data obtained from the ITC assay, both of the human proteins (HLY and HZF) were demonstrated to bind to their respective SAG1 and SAG2 proteins.

Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-Tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic-Targeting and Imaging.

Matrix metalloproteinases (MMPs) activatable imaging probe has been explored for tumor detection. However, activation of the probe is mainly done in the extracellular space without intracellular uptake of the probe for more sensitivity. Although cell-penetrating peptides (CPPs) have been demonstrated to enable intracellular delivery of the imaging probe, they nevertheless encounter off-target delivery of the cargos to normal tissues. Herein, we have developed a dual MMP-2-activatable and tumor cell-permeable magnetic nanoprobe to simultaneously achieve selective and intracellular tumor imaging. This novel imaging probe was constructed by self-assembling a hexahistidine-tagged (His-tagged) fluorescent fusion protein chimera and nickel ferrite nanoparticles via a chelation mechanism. The His-tagged fluorescent protein chimera consisted of a red fluorescent protein mCherry that acted as the fluorophore, the low-molecular-weight protamine peptide as the CPP, and the MMP-2 cleavage sequence fused with the hexahistidine tag, whereas the nickel ferrite nanoparticles functioned as the His-tagged protein binder and also the fluorescent quencher. Both in vitro and in vivo results revealed that this imaging probe would not only remain nonpermeable to normal tissues, thereby offsetting the nonselective cellular uptake, but was also suppressed of fluorescent signals during magnetic tumor-targeting in the circulation, primarily because of the masking of the CPP activity and quenching of the fluorophore by the associated NiFe2O4 nanoparticles. However, these properties were recovered or "turned on" by the action of tumor-associated MMP-2 stimuli, leading to cell penetration of the nanoprobes as well as fluorescence restoration and visualization within the tumor cells. In this regard, the presented tumor-activatable and cell-permeable system deems to be an appealing platform to achieve selective tumor imaging and intracellular protein delivery. Its impact is therefore significant, far-reaching, and wide-spread.

Characterization and expression patterns of mannose-binding lectin (MMBL) gene in mulberry (Morus multicaulis) and its prokaryotic expression in E. coli

Mannose-binding lectin (MBL) is an important natural immune molecule which plays a significant role in plant self-defense and shows a broad spectrum resistance on plant pathogens, viral pathogens, and fungi. In the present paper, a cDNA sequence encoding MBL, which was designated MMBL (GenBank accession NO: KY348866), was cloned from the leaves of mulberry (Morus multicaulis) based on mulberry expressed sequence tags (EST) and homologous cloning technology using RT-PCR. The cDNA was 978bp in length with a 5′ untranslated regions (UTR) of 189bp, a 3′ UTR of 303bp and an open reading frame (ORF) of 486bp encoding a protein of 161 amino acids. The estimated molecular weight and isoelectric point (pI) of the putative protein were16.97kDa and 6.07, respectively. The MMBL had Jacalin domain and six sugar binding sites, and belonged to Jacalin-like superfamily. Phylogenetic analysis based on the amino acid sequences encoded by the MBL gene from various species showed that mulberry was closely related to Morus notabilis, Ipomoea nil, Capsicum annuum, Solanum lycopersicum and Solanum tuberosum. Quantitative real-time PCR (qRT-PCR) analysis revealed that MMBL was expressed in all the tested tissues, including leaf, bud, fruit, stem, phloem and xylem of the mulberry with the highest expression in the bud and leaf. The expression level of the mRNA has changed significantly under drought, cold, salt and mechanical damage stress treatments compared to the normal growth environment. The ORF segment of the MMBL was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. SDS-PAGE and western blot results revealed that His-tagged fusion protein was successfully expressed. Overall, these results showed a better understanding of the molecular basis for the signal transduction mechanism during the stress responses in mulberry trees.

Humoral and cytokine response elicited during immunisation with recombinant Immune Mapped protein-1 (EtIMP-1) and oocysts of Eimeria tenella

Eimeria tenella, the causative agent of caecal coccidiosis, is a pathogenic gut dwelling protozoan which can cause severe morbidity and mortality in farmed chickens. Immune mapped protein-1 (IMP-1) has been identified as an anticoccidial vaccine candidate; in the present study allelic polymorphism was assessed across the IMP-1 coding sequence in E. tenella isolates from four countries and compared with the UK reference Houghton strain. Nucleotide diversity was low, limited to expansion/contraction of a CAG triplet repeat and five substitutions, three of which were non-synonymous. The EtIMP-1 coding sequence from a cloned Indian E. tenella isolate was expressed in E. coli and purified as a His-tagged thioredoxin fusion protein. An in-vivo vaccination and challenge trial was conducted to test the vaccine potential of recombinant EtIMP-1 (rEtIMP-1) and to compare post-vaccination immune responses of chickens to those stimulated by live oocyst infection. Following challenge, parasite replication measured using quantitative PCR was significantly reduced in chickens that had been vaccinated with rEtIMP-1 (rIC group; 67% reduction compared to UC or unimmunised controls; 79% reduction compared to rTC group or recombinant thioredoxin mock-immunised controls, p<0.05), or the birds vaccinated by infection with oocysts (OC group, 90% compared to unimmunised controls). Chickens vaccinated with oocysts (OC) had significantly higher levels of interferon gamma in their serum post-challenge, compared to rEtIMP-1 vaccinated birds (rIC). Conversely rEtIMP-1 (rIC) vaccinated birds had significantly higher antigen specific serum IgY responses, correlating with higher serum IL-4 (both p<0.05).

Novel ribonuclease activity of cusativin from Cucumis sativus for mapping nucleoside modifications in RNA.

A recombinant ribonuclease, cusativin, was characterized for its cytidine-specific cleavage ability of RNA to map chemical modifications. Following purification of native cusativin protein as described before (Rojo et al. Planta 194:328, 17), partial amino acid sequencing was carried out to identify the corresponding protein coding gene in cucumber genome. Cloning and heterologous expression of the identified gene in Escherichia coli resulted in successful production of active protein as a C-terminal His-tag fusion protein. The ribonuclease activity and cleavage specificity of the fusion protein were confirmed with a variety of tRNA isoacceptors and total tRNA. Characterization of cusativin digestion products by ion-pairing reverse-phase liquid chromatography coupled with mass spectrometry (IP-RP-LC-MS) analysis revealed cleavage of CpA, CpG, and CpU phosphodiester bonds at the 3'-terminus of cytidine under optimal digestion conditions. Ribose methylation or acetylation of cytosine inhibited RNA cleavage. The CpC phosphodiester bond was also resistant to cusativin-mediated RNA cleavage; a feature to our knowledge has not been reported for other nucleobase-specific ribonucleases. Here, we demonstrate the analytical utility of such a novel feature for obtaining high-sequence coverage and accurate mapping of modified residues in substrate RNAs. Graphical abstract Cytidine-specific novel ribonuclease activity of cusativin.