Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C1175175 (SARS)
 19,188 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Procedures for synthesizing alpha-amino acids on a chip for coordination with transitional metal ions and a His-Tagged protein have been successfully developed as a stable protein microarray. Using the recombinant His-Tagged 3CL-protease (3CLpro) as a model for attachment to chips containing D-/L-Glu, Asp, Orn, Ser via different transitional metal ions, it was found that the Orn chip was the best of affinity binding and stability by which Zn2+ was the best metal ion for affinity while Co2+ was the best metal ion for stability. Thus, this protein microarray can be alternatively used as a high throughput screening method for rapid detection against SARS CoV 3CLpro and/or efficient purification of other Tagged proteins.
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PMID:Protein microarray using alpha-amino acids as metal tags on chips. 1571 98

This article describes the pathological studies of fatal severe acute respiratory syndrome (SARS) in a 73-year-old man during an outbreak of SARS in Taiwan, 2003. Eight days before onset of symptoms, he visited a municipal hospital that was later identified as the epicenter of a large outbreak of SARS. On admission to National Taiwan University Hospital in Taipei, the patient experienced chest tightness, progressive dyspnea, and low-grade fever. His condition rapidly deteriorated with increasing respiratory difficulty, and he died 7 days after admission. The most prominent histopathologic finding was diffuse alveolar damage of the lung. Immunohistochemical and in situ hybridization assays demonstrated evidence of SARS-associated coronavirus (SARS-CoV) infection in various respiratory epithelial cells, predominantly type II pneumocytes, and in alveolar macrophages in the lung. Electron microscopic examination also revealed coronavirus particles in the pneumocytes, and their identity was confirmed as SARS-CoV by immunogold labeling electron microscopy. This report is the first to describe the cellular localization of SARS-CoV in human lung tissue by using a combination of immunohistochemistry, double-stain immunohistochemistry, in situ hybridization, electron microscopy, and immunogold labeling electron microscopy. These techniques represent valuable laboratory diagnostic modalities and provide insights into the pathogenesis of this emerging infection.
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PMID:Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan. 1579 76

A new Coronavirus (SARS-CoV) is the aetiological agent of the severe acute respiratory syndrome (SARS). Because of the critical role played by serological assays for SARS diagnosis, an in-house ELISA based on SARS-CoV recombinant antigens was developed. The SARS-CoV nucleocapsid protein (N), three N fragments (N1, N2, and N3) and the intraviral domain of the membrane protein (M2) were cloned and expressed in Escherichia coli as histidine-tagged proteins. Six reference sera from SARS patients were used to detect virus-specific IgG in an ELISA using each recombinant protein as coating antigen. High-titre positive reactions were detected in all SARS positive sera. The specificity of the assay appears to be high as no positive reaction was detected in the sera of 20 healthy subjects and 73 patients with non-SARS, low-tract respiratory infections. Specific hyper-immune sera to SARS-CoV and the recombinant proteins, N, N1, N2, N3, and M2 were also generated in mice and rabbits. The specificity of these sera was confirmed by an immunocytochemical assay on biochips of SARS-CoV infected and uninfected cells.
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PMID:Recombinant protein-based ELISA and immuno-cytochemical assay for the diagnosis of SARS. 1583 68

The SARS-nsp13 protein was identified as an mRNA cap1 methyltransferase. In this study, the nsp13 gene was cloned from the SARS-CoV PUMC02 strain viral RNA by RT-PCR, and inserted into the expression plasmid pET30a(+). The recombinant plasmid pET30a(+)-nsp13 was confirmed by restriction enzymes and sequencing analysis, and transformed into Escherichia coli BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5mM IPTG and purified by a single Ni(2+) affinity chromatography. The protein was validated by western blot and MS analysis. A large quantity of the nsp13 protein obtained with this method may be useful for further study of its structure and function.
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PMID:Molecular cloning, expression, and purification of SARS-CoV nsp13. 1586 8

Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.
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PMID:Expression and purification of SARS coronavirus proteins using SUMO-fusions. 1593 95

Viral proteases are essential for pathogenesis and virulence of severe acute respiratory syndrome coronavirus (SARS-CoV). Little information is available on SARS-CoV papain-like protease 2 (PLP2), and development of inhibitors against PLP2 is attractive for antiviral therapy. Here, we report the characterization of SARS-CoV PLP2 (from residues 1414 to 1858) purified from baculovirus-infected insect cells. We demonstrate that SARS-CoV PLP2 by itself differentially cleaves between the amino acids Gly180 and Ala181, Gly818 and Ala819, and Gly2740 and Lys2741 of the viral polypeptide pp1a, as determined by reversed-phase high-performance liquid chromatography analysis coupled with mass spectrometry. This protease is especially selective for the P1, P4, and P6 sites of the substrate. The study demonstrates, for the first time among coronaviral PLPs, that the reaction mechanism of SARS-CoV PLP2 is characteristic of papain and compatible with the involvement of the catalytic dyad (Cys)-S(-)/(His)-Im(+)H ion pair. With a fluorogenic inhibitor-screening platform, we show that zinc ion and its conjugates potently inhibit the enzymatic activity of SARS-CoV PLP2. In addition, we provided evidence for evolutionary reclassification of SARS-CoV. The results provide important insights into the biochemical properties of the coronaviral PLP family and a promising therapeutic way to fight SARS-CoV.
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PMID:Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, purification, characterization, and inhibition. 1604 12

Histidine-tagged N (rNH) and E (rEH) proteins of Severe Acute Respiratory Syndrome (SARS)-coronovirus were expressed in the baculovirus/insect cell system and purified by immobilized metal affinity chromatography. rNH and rEH proteins differed markedly with respect to expression levels, cell death kinetics and subcellular localizations that led to different extraction and purification schemes. The features of both proteins are compared and the potential applications of purified rNH and rEH are discussed.
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PMID:Expression and purification of N and E proteins from severe acute respiratory syndrome (SARS)-associated coronavirus: a comparative study. 1609 81

The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.
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PMID:[Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity]. 1617 89

Replication of the approximately 30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.
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PMID:ADP-ribose-1"-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture. 1618 75

The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-dependent endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration, while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low endoribonuclease activity, suggesting that oligomerization is required for endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites.
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PMID:Mutational analysis of the SARS virus Nsp15 endoribonuclease: identification of residues affecting hexamer formation. 1621 69


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