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Group 21 and 5 allergens are homologous house dust mite proteins known as mid-tier allergens. To reveal the biological function of group 21 allergens and to understand better the allergenicity of the rDer f 21 allergen, we determined the 1.5Å crystal structure of rDer f 21 allergen from Dermatophagoides farinae. The rDer f 21 protein consists of a three helical bundle, similar to available structures of group 21 and homologous group 5 allergens. The rDer f 21 dimer forms a hydrophobic binding pocket similar to the one in the Der p 5 allergen, which indicates that both of the homologous groups could share a similar function. By performing structure-guided mutagenesis, we mutated all 38 surface-exposed polar residues of the rDer f 21 allergen and carried out immuno-dot blot assays using 24 atopic sera. Six residues, K10, K26, K42, E43, K46, and K48, which are located in the region between the N-terminus and the loop 1 of rDer f 21 were identifed as the major IgE epitopes of rDer f 21. Epitope mapping of all potential IgE epitopes on the surface of the rDer f 21 crystal structure revealed heterogeneity in the sIgE recognition of the allergen epitopes in atopic individuals. The higher the allergen-sIgE level of an individual, the higher the number of epitope residues that are found in the allergen. The results illustrate the clear correlation between the number of specifc major epitope residues in an allergen and the sIgE level of the atopic population.

Group 21 and 5 allergens are homologous house dust mite proteins known as mid-tier allergens. To reveal the biological function of group 21 allergens and to understand better the allergenicity of the rDer f 21 allergen, we determined the 1.5Å crystal structure of rDer f 21 allergen from Dermatophagoides farinae. The rDer f 21 protein consists of a three helical bundle, similar to available structures of group 21 and homologous group 5 allergens. The rDer f 21 dimer forms a hydrophobic binding pocket similar to the one in the Der p 5 allergen, which indicates that both of the homologous groups could share a similar function. By performing structure-guided mutagenesis, we mutated all 38 surface-exposed polar residues of the rDer f 21 allergen and carried out immuno-dot blot assays using 24 atopic sera. Six residues, K10, K26, K42, E43, K46, and K48, which are located in the region between the N-terminus and the loop 1 of rDer f 21 were identifed as the major IgE epitopes of rDer f 21. Epitope mapping of all potential IgE epitopes on the surface of the rDer f 21 crystal structure revealed heterogeneity in the sIgE recognition of the allergen epitopes in atopic individuals. The higher the allergen-sIgE level of an individual, the higher the number of epitope residues that are found in the allergen. The results illustrate the clear correlation between the number of specifc major epitope residues in an allergen and the sIgE level of the atopic population.

Proteins of the DUF866 superfamily are exclusively found in eukaryotic cells. A member of the DUF866 superfamily, C1ORF123, is a human protein found in the open reading frame 123 of chromosome 1. The physiological role of C1ORF123 is yet to be determined. The only available protein structure of the DUF866 family shares just 26% sequence similarity and does not contain a zinc binding motif. Here, we present the crystal structure of the recombinant human C1ORF123 protein (rC1ORF123). The structure has a 2-fold internal symmetry dividing the monomeric protein into two mirrored halves that comprise of distinct electrostatic potential. The N-terminal half of rC1ORF123 includes a zinc-binding domain interacting with a zinc ion near to a potential ligand binding cavity. Functional studies of human C1ORF123 and its homologue in the fission yeast Schizosaccharomyces pombe (SpEss1) point to a role of DUF866 protein in mitochondrial oxidative phosphorylation.

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp280, Leu294 and Ala303). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily.

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp280, Leu294 and Ala303). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily.

Proteins of the DUF866 superfamily are exclusively found in eukaryotic cells. A member of the DUF866 superfamily, C1ORF123, is a human protein found in the open reading frame 123 of chromosome 1. The physiological role of C1ORF123 is yet to be determined. The only available protein structure of the DUF866 family shares just 26% sequence similarity and does not contain a zinc binding motif. Here, we present the crystal structure of the recombinant human C1ORF123 protein (rC1ORF123). The structure has a 2-fold internal symmetry dividing the monomeric protein into two mirrored halves that comprise of distinct electrostatic potential. The N-terminal half of rC1ORF123 includes a zinc-binding domain interacting with a zinc ion near to a potential ligand binding cavity. Functional studies of human C1ORF123 and its homologue in the fission yeast Schizosaccharomyces pombe (SpEss1) point to a role of DUF866 protein in mitochondrial oxidative phosphorylation.

C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å3 Da−1 suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.

C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å3 Da−1 suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.

Melioidosis is an infectious disease caused by the pathogenic bacterium Burkholderia pseudomallei. Whole-genome sequencing revealed that the B. pseudomallei genome includes 5855 coding DNA sequences (CDSs), of which ∼25% encode hypothetical proteins. A pathogen-associated hypothetical protein, BPSL1038, was overexpressed in Escherichia coli, purified and crystallized using vapour-diffusion methods. A BPSL1038 protein crystal that grew using sodium formate as precipitant diffracted to 1.55 Å resolution. It belonged to space group C2221, with unit-cell parameters a = 85.36, b = 115.63, c = 46.73 Å. The calculated Matthews coefficient (VM) suggests that there are two molecules per asymmetric unit, with a solvent content of 48.8%.

Melioidosis is an infectious disease caused by the pathogenic bacterium Burkholderia pseudomallei. Whole-genome sequencing revealed that the B. pseudomallei genome includes 5855 coding DNA sequences (CDSs), of which ∼25% encode hypothetical proteins. A pathogen-associated hypothetical protein, BPSL1038, was overexpressed in Escherichia coli, purified and crystallized using vapour-diffusion methods. A BPSL1038 protein crystal that grew using sodium formate as precipitant diffracted to 1.55 Å resolution. It belonged to space group C2221, with unit-cell parameters a = 85.36, b = 115.63, c = 46.73 Å. The calculated Matthews coefficient (VM) suggests that there are two molecules per asymmetric unit, with a solvent content of 48.8%.

Renin is an important honnone in kidney regulating the renin-angiotensin system (RAS); which plays an important role in human blood pressure. Renin is a highly specific endopeptidase cleaving the Leu-Leu bond in angiotensinogen to generate angiotensin I. Recently, renin was found in organs other than the kidney such as adrenal, ovary, testis, uterus, placenta, anterior pituitary and brain, implicating its involvement in the regulation of numerous activities. Prorenin is the inactive precursor of the renin which regulates the blood pressure and electrolyte balance. Prorenin can be activated in vitro following nonproteolysis and proteolysis. The isolation of prorenin or renin from organs including kidney is extremely difficult due to its very low concentration and its instability. Therefore, recombinant protein technologies are used to produce the recombinant human renin protein. In this study, the full-length human renin coding gene (REN) was isolated from the human kidney cDNA library by using the polymerase chain reaction (PCR) technique. The primers (RF1 & RR1) used were designed based on the human mRNA renin gene sequence from GenBank [gi |4506474| ref | NM_000537.1|]. The PCR amplified REN gene was cloned into pCR-Blunt cloning vector. Sequencing was carried out and the result shows 99.9% identical to the published sequence. The REN gene was cloned into two different E. coli expression vectors, pRSETB and pGEX4T l , to express the recombinant protein. Construct pRB-R was successfully expressed in E. coli strains BL2 1-S1 and BL21 (DE3)pLysS with the recombinant protein corresponding to the expected size -48 kDa. Construct pGT-R was expressed in BL2 1 (DE3)pLysS with the size -66 kDa. Both recombinant proteins have been confirmed with western blotting by using monoclonal anti-His antibody (recombinant protein derived from pRSET vector) and monoclonal anti-GST antibody (recombinant protein derived from p GEX4Tl vector). The result of the expression shows that the

Renin is an important honnone in kidney regulating the renin-angiotensin system (RAS); which plays an important role in human blood pressure. Renin is a highly specific endopeptidase cleaving the Leu-Leu bond in angiotensinogen to generate angiotensin I. Recently, renin was found in organs other than the kidney such as adrenal, ovary, testis, uterus, placenta, anterior pituitary and brain, implicating its involvement in the regulation of numerous activities. Prorenin is the inactive precursor of the renin which regulates the blood pressure and electrolyte balance. Prorenin can be activated in vitro following nonproteolysis and proteolysis. The isolation of prorenin or renin from organs including kidney is extremely difficult due to its very low concentration and its instability. Therefore, recombinant protein technologies are used to produce the recombinant human renin protein. In this study, the full-length human renin coding gene (REN) was isolated from the human kidney cDNA library by using the polymerase chain reaction (PCR) technique. The primers (RF1 & RR1) used were designed based on the human mRNA renin gene sequence from GenBank [gi |4506474| ref | NM_000537.1|]. The PCR amplified REN gene was cloned into pCR-Blunt cloning vector. Sequencing was carried out and the result shows 99.9% identical to the published sequence. The REN gene was cloned into two different E. coli expression vectors, pRSETB and pGEX4T l , to express the recombinant protein. Construct pRB-R was successfully expressed in E. coli strains BL2 1-S1 and BL21 (DE3)pLysS with the recombinant protein corresponding to the expected size -48 kDa. Construct pGT-R was expressed in BL2 1 (DE3)pLysS with the size -66 kDa. Both recombinant proteins have been confirmed with western blotting by using monoclonal anti-His antibody (recombinant protein derived from pRSET vector) and monoclonal anti-GST antibody (recombinant protein derived from p GEX4Tl vector). The result of the expression shows that the

Renin is an important honnone in kidney regulating the renin-angiotensin system (RAS); which plays an important role in human blood pressure. Renin is a highly specific endopeptidase cleaving the Leu-Leu bond in angiotensinogen to generate angiotensin I. Recently, renin was found in organs other than the kidney such as adrenal, ovary, testis, uterus, placenta, anterior pituitary and brain, implicating its involvement in the regulation of numerous activities. Prorenin is the inactive precursor of the renin which regulates the blood pressure and electrolyte balance. Prorenin can be activated in vitro following nonproteolysis and proteolysis. The isolation of prorenin or renin from organs including kidney is extremely difficult due to its very low concentration and its instability. Therefore, recombinant protein technologies are used to produce the recombinant human renin protein. In this study, the full-length human renin coding gene (REN) was isolated from the human kidney cDNA library by using the polymerase chain reaction (PCR) technique. The primers (RF1 & RR1) used were designed based on the human mRNA renin gene sequence from GenBank [gi |4506474| ref | NM_000537.1|]. The PCR amplified REN gene was cloned into pCR-Blunt cloning vector. Sequencing was carried out and the result shows 99.9% identical to the published sequence. The REN gene was cloned into two different E. coli expression vectors, pRSETB and pGEX4T l , to express the recombinant protein. Construct pRB-R was successfully expressed in E. coli strains BL2 1-S1 and BL21 (DE3)pLysS with the recombinant protein corresponding to the expected size -48 kDa. Construct pGT-R was expressed in BL2 1 (DE3)pLysS with the size -66 kDa. Both recombinant proteins have been confirmed with western blotting by using monoclonal anti-His antibody (recombinant protein derived from pRSET vector) and monoclonal anti-GST antibody (recombinant protein derived from p GEX4Tl vector). The result of the expression shows that the