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The purpose of this study was to provide a new insight on the response of canines to stress exposure; the ionomic profiles of canine hair (2.8 ± 0.3 years, 15.17 ± 2.1 kg) (n = 10) was determined before and after lipopolysaccharide (LPS) injections. LPS was intramuscularly injected to induce inflammatory stress responses which were confirmed by observing increases in the level of serum cortisol, aldosterone, and inflammatory cytokines such as IL-6, IL-1β, and TNF-α. The hair contents of 17 elements were obtained by applying analytical procedures using the inductively coupled plasma mass spectrometry (ICP-MS). The following elements: sodium(Na) and potassium(K) among macro-elements, iron(Fe) and manganese(Mn) among micro-elements, and aluminum(Al), nickel(Ni), and lead(Pb) for toxic elements, showed significant increased levels with the immunological stress. The degree of increase in toxic elements was remarkable with the stress exposure. A forty-five-fold increase seen in Al accumulation with the stress exposure was noteworthy. Although mercury(Hg) and cadmium(Cd) showed decreased levels with the stress exposure, the degree was negligible compared to the level of increase. Correlation pattern between the elements was changed with the immunological stress. Toxic elements became more correlated with macro- or micro-elements than with toxic elements themselves after the stress exposure. Principal component analysis (PCA) showed that LPS challenge shifted the overall hair mineral profiles to a consistent direction changing Al and K up, even in animals with different hair mineral profiles before LPS treatment. In conclusion, the multivariate data processing and study of element distribution patterns provided new information about the ionomic response of the canine hairs to immunological stress, i.e., the ionomic profiles of canine hairs is strongly affected by the stress induced by LPS injections.

A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homogeneity in two steps employing cation and an anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The optimal activity occurred at pH 5.5 and 40 degrees C. The Michaelis constant (Km) and maximum reaction rate (Vmax) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively. The enzyme was strongly inhibited by Cu2+, Cd2+, Mn2+, and ethylenediaminetetraacetic acid (EDTA). It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates. The enzyme efficiently released orthophosphate from wheat bran and soybean meal.

BackgroundPorcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which therefore is an ideal target for the development of subunit vaccine. In an attempt to develop a subunit vaccine for PEDV, we cloned two different fragments of S protein and expressed as glutathione S-transferase (GST)-tagged fusion proteins, namely rGST-COE and rGST-S1D, in E.coli. However, the expression of these recombinant protein antigens using a variety of expression vectors, strains, and induction conditions invariably resulted in inclusion bodies. To achieve the soluble expression of recombinant proteins, several chaperone co-expression systems were tested in this study.ResultsWe firstly tested various chaperone co-expression systems and found that co-expression of trigger factor (TF) with recombinant proteins at 15 °C was most useful in soluble production of rGST-COE and rGST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble rGST-COE and rGST-S1D were purified using glutathione Sepharose 4B with a yield of 7.5 mg/l and 5 mg/l, respectively. Purified proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. In an indirect ELISA, purified proteins showed immune reactivity with pig anti-PEDV immune sera. Finally, immunization of mice with 10 μg of purified proteins elicited highly potent serum IgG and serum neutralizing antibody titers.ConclusionsIn this study, soluble production of recombinant spike protein of PEDV, rGST-COE and rGST-S1D, were achieved by using TF chaperone co-expression system. Our results suggest that soluble rGST-COE and rGST-S1D produced by co-expressing chaperones may have the potential to be used as subunit vaccine antigens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0268-7) contains supplementary material, which is available to authorized users.

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-β-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) DH5α. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli DH5α harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine–Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was 55°C, but it retained over 90% of maximum activity in a broad temperature range (40°C to 60°C). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, Km and Vmax of rEG1 were 0.39% CMC and 143 U/mg, respectively.