Isolation, Cloning and Expression of Recombinant Human Renin in Escherichia Coli System

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 combination of the expression vector pRSETB and host BL2 1 (DE3)pLysS gave the highest soluble fraction of recombinant protein

Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerol-specific enzyme for citral biosynthesis

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

Fenazin sebagai potensi antibiotik baru daripada Streptomyces kebangsaanensis

Fenazin merupakan metabolit sekunder yang biasanya disintesis secara semula jadi oleh Pseudomonas dan Streptomyces. Ia merupakan sebatian heterosiklik yang mempunyai sebatian bernitrogen pada struktur teras cecincin. Kajian mengenai antibiotik ini telah bermula seawal abad ke-19 lagi dan ternyata menjadi calon dadah yang berpotensi tinggi dalam dunia perubatan. Sehingga kini, lebih daripada 100 jenis fenazin telah diterokai daripada sumber semula jadi dan boleh bertindak sebagai antibakteria, antikanser, antivirus, antitumor serta antiparasit. Setakat ini, kajian biosintesis fenazin yang telah dijalankan terhadap Pseudomonas dan Streptomyces telah mendedahkan gen yang bertanggungjawab dalam tapak jalan biosintesis fenazin, namun begitu, gen khusus yang terlibat dalam penghasilan terbitan fenazin yang kompleks masih dalam hipotesis. Dalam ulasan ini, kami membincangkan kepentingan fenazin serta pemahaman terkini tentang tapak jalan biosintesis fenazin yang berjaya diterokai di dalam Streptomyces kebangsaanensis

Crystallization and X-ray crystallographic analysis of recombinant TylP, a putative γ-butyrolactone receptor protein from Streptomyces fradiae

TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å

Crystallization and preliminary crystallographic studies of the hypothetical protein BPSL 1038 from Burkholderia pseudomallei

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%

Structural investigation of proteins from the exosome superoperon of M. thermautotrophicus

The exosome is a 3' to 5' RNA-processing and RNA-degrading macromolecular assembly. The genes encoding exosome core proteins are conserved in ar~haeal and eukaryotic cells. The work presented in this thesis is based on a structural and functional approach, using. protein crystallography, yeast two-hybrid experiments and EMSA assays, to study several proteins encoded in the exosOIne superoperon of Methanothermobacter thermautotrophicus (Mth). Crystal structures were determined for the gene products Mth680, Mth685, Mth689 and the exosome core, which consists of proteins encoded by the Mth682 and Mth683 genes. Research work was also carried out to identify protein interaction partners through protein-protein and protein-RNA interaction assays. The Mth680 protein is a member of the Imp4/Brix superfamily, which in eukaryotes is involved in ribosome biogenesis. We named this protein Mil for Mth Imp4.-Like. Its Xray structure provided the first information about the fold of proteins belonging to the Imp4/Brix superfamily. The structure revealed that the N-terminal and C-tenninal halves of this protein have very similar folds suggesting that the superfamily evolved by gene duplication. The fold of each half is similar to the fold of anticodon binding domain of class IIa aminoacyl-tRNA synthetases. A yeast-two hybrid experiment has identified the Mthl215 protein (homologue of the yeast Nopl protein) as a possible interaction partner of Mil. The binding of Mil to the exosome core has also been detected. Finally, Mil was also found to interact with single stranded RNA sequences. The Mth685 protein is an orthologue of the human Shwachman-Bodian-Diamond Syndrome protein (SBDS) therefore we named it mthSBDS. The structure of mthSBDS reveals a high degree of flexibility between the domains of this protein which are arranged in an 'L' shape. Several possible mthSBDS protein interaction partners including sensory transduction histidine kinase, alanyl tRNA synthetase: adenosylmethionine synthetase, Glutamate N-acetyltransferase and ribosomal proteins ofLl, L2, and Ll4 were identified. The function of Mth689 protein remains unknown. Its crystal structure reveals similarity to the large ribosomal subunit protein L5. Analysis of Mth689 structure reveals the presence of a positively charged concave surface that may interact with nucleic acids. I have also detefII}ined the X-ray structure of the Mth exosome core assembly that I prepared by co-expressing Mth682 and Mth683 proteins. The structure shows that the architechture of the active site and the positions of all active site residues are conserved, suggesting a similar phosphorolytic mechanism proposed for exosmes from other organisms. The structure shows for the fIrst time the presence of catalytically critical inorganic phosphate ions in each phosphorolytic active site further supporting the mechanism proposed earlier.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Crystal structure of Mil (Mth680): internal duplication and similarity between the Imp4/Brix domain and the anticodon-binding domain of class IIa aminoacyl-tRNA synthetases

Proteins of the Imp4/Brix superfamily are involved in ribosomal RNA processing, an essential function in all cells. We report the first structure of an Imp4/Brix superfamily protein, the Mil (for Methanothermobacter thermautotrophicus Imp4-like) protein (gene product Mth680), from the archaeon M. thermautotrophicus. The amino- and carboxy-terminal halves of Mil show significant structural similarity to one another, suggesting an origin by means of an ancestral duplication. Both halves show the same fold as the anticodon-binding domain of class IIa aminoacyl-tRNA synthetases, with greater conservation seen in the N-terminal half. This structural similarity, together with the charge distribution in Mil, suggests that Imp4/Brix superfamily proteins could bind single-stranded segments of RNA along a concave surface formed by the N-terminal half of their β-sheet and a central α-helix. The crystal structure of Mil is incompatible with the presence, in the Imp4/Brix domain, of a helix–turn–helix motif that was proposed to comprise the RNA-binding moiety of the Imp4/Brix proteins

Whole-genome shotgun sequence of phenazine-producing endophytic Streptomyces kebangsaanensis SUK12

Streptomyces sp. produces bioactive compounds with a broad spectrum of activities. Streptomyces kebangsaanesis SUK12 has been identified as a novel endophytic bacteria isolated from ethnomedicinal plant Portulaca olerace, and was found to produce the phenazine class of biologically active antimicrobial metabolites. The potential use of the phenazines has led to our research interest in determining the genome sequence of Streptomyces kebangsaanensis SUK12. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number PRJNA269542. The raw sequence data are available [https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP105770]

IgE-binding residues analysis of the house dust mite allergen Der p 23

10.1038/s41598-020-79820-ySCIENTIFIC REPORTS11