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1.
Artigo em Inglês | MEDLINE | ID: mdl-37817332

RESUMO

Background: Toxigenic Corynebacterium ulcerans is an emerging zoonosis globally, causing both cutaneous and respiratory diphtheria-like illness. In Queensland, human infection with toxigenic C. ulcerans is rare, with only three cases reported before October 2015. This case series describes five subsequent cases of toxigenic C. ulcerans in Queensland with links to companion animals. Methods: All data were collected as part of routine public health response, and strains were whole genome sequenced for further characterisation. Household contacts were screened, treated with appropriate antibiotics, and received a diphtheria toxoid-containing vaccine if more than five years had elapsed since their last dose. Findings: No epidemiological or genomic links could be established between any of the five patients, including between the two cases notified from the same locality within eight days of each other. The C. ulcerans strains from Cases Two, Four and Five were closely related to the strains isolated from their respective pets by whole genome sequencing. Domestic dogs were identified as the most likely mode of transmission for Cases One and Three; however, this was unable to be laboratory confirmed, since Case One's dog was treated with antibiotics before it could be tested, and Case Three's dog was euthanised and cremated prior to case notification. Interpretation: These are the first reported Australian cases of this emerging zoonosis with links to companion animals. These cases demonstrate the likely transmission route between companion animals and humans, with no evidence of human-to-human transmission. The existing requirement in the Queensland Health Public Health Management Guidelines, of restrictions on cases and some contacts while awaiting swab results, is currently under review.


Assuntos
Infecções por Corynebacterium , Difteria , Humanos , Animais , Cães , Infecções por Corynebacterium/tratamento farmacológico , Infecções por Corynebacterium/epidemiologia , Infecções por Corynebacterium/veterinária , Queensland/epidemiologia , Austrália/epidemiologia , Difteria/tratamento farmacológico , Difteria/epidemiologia , Difteria/microbiologia , Zoonoses/epidemiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico
2.
Nat Commun ; 14(1): 1051, 2023 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-36828918

RESUMO

A new variant of Streptococcus pyogenes serotype M1 (designated 'M1UK') has been reported in the United Kingdom, linked with seasonal scarlet fever surges, marked increase in invasive infections, and exhibiting enhanced expression of the superantigen SpeA. The progenitor S. pyogenes 'M1global' and M1UK clones can be differentiated by 27 SNPs and 4 indels, yet the mechanism for speA upregulation is unknown. Here we investigate the previously unappreciated expansion of M1UK in Australia, now isolated from the majority of serious infections caused by serotype M1 S. pyogenes. M1UK sub-lineages circulating in Australia also contain a novel toxin repertoire associated with epidemic scarlet fever causing S. pyogenes in Asia. A single SNP in the 5' transcriptional leader sequence of the transfer-messenger RNA gene ssrA drives enhanced SpeA superantigen expression as a result of ssrA terminator read-through in the M1UK lineage. This represents a previously unappreciated mechanism of toxin expression and urges enhanced international surveillance.


Assuntos
Escarlatina , Infecções Estreptocócicas , Humanos , Streptococcus pyogenes/genética , Escarlatina/epidemiologia , Superantígenos , Proteínas de Bactérias/genética , Reino Unido , Exotoxinas/genética , Mutação , Austrália
3.
Artigo em Inglês | MEDLINE | ID: mdl-31091405

RESUMO

In September 2016, an invasive group A streptococcal disease outbreak occurred among residents of a residential aged care facility. An expert advisory group recommended mass prophylaxis for residents and staff in addition to strict infection control practices to prevent further spread. Whole genome sequencing confirmed the cases were related.


Assuntos
Antibioticoprofilaxia , Surtos de Doenças/prevenção & controle , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/isolamento & purificação , Idoso , Instituição de Longa Permanência para Idosos , Humanos , Controle de Infecções , Casas de Saúde , Filogenia , Polimorfismo de Nucleotídeo Único , Saúde Pública , Queensland/epidemiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/genética , Sequenciamento Completo do Genoma
4.
Front Microbiol ; 8: 603, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28428781

RESUMO

Listeriosis remains among the most important bacterial illnesses, with a high associated mortality rate. Efforts to control listeriosis require detailed knowledge of the epidemiology of the disease itself, and its etiological bacterium, Listeria monocytogenes. In this study we provide an in-depth analysis of the epidemiology of 224 L. monocytogenes isolates from Australian clinical and non-clinical sources. Non-human sources included meat, dairy, seafood, fruit, and vegetables, along with animal and environmental isolates. Serotyping, Multi-Locus Sequence Typing, and analysis of inlA gene sequence were performed. Serogroups IIA, IIB, and IVB comprised 94% of all isolates, with IVB over-represented among clinical isolates. Serogroup IIA was the most common among dairy and meat isolates. Lineage I isolates were most common among clinical isolates, and 52% of clinical isolates belonged to ST1. Overall 39 STs were identified in this study, with ST1 and ST3 containing the largest numbers of L. monocytogenes isolates. These STs comprised 40% of the total isolates (n = 90), and both harbored isolates from clinical and non-clinical sources. ST204 was the third most common ST. The high prevalence of this group among L. monocytogenes populations has not been reported outside Australia. Twenty-seven percent of the STs in this study contained exclusively clinical isolates. Analysis of the virulence protein InlA among isolates in this study identified a truncated form of the protein among isolates from ST121 and ST325. The ST325 group contained a previously unreported novel mutation leading to production of a 93 amino acid protein. This study provides insights in the population structure of L. monocytogenes isolated in Australia, which will contribute to public health knowledge relating to this important human pathogen.

5.
Emerg Infect Dis ; 23(1): 105-107, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27983494

RESUMO

By conducting a molecular characterization of Corynebacterium diphtheriae strains in Australia, we identified novel sequences, nonfunctional toxin genes, and 5 recent cases of toxigenic cutaneous diphtheria. These findings highlight the importance of extrapharyngeal infections for toxin gene-bearing (functional or not) and non-toxin gene-bearing C. diphtheriae strains. Continued surveillance is recommended.


Assuntos
Corynebacterium diphtheriae/patogenicidade , Toxina Diftérica/genética , Difteria/epidemiologia , Genes Bacterianos , Austrália/epidemiologia , Corynebacterium diphtheriae/genética , Difteria/imunologia , Difteria/microbiologia , Difteria/prevenção & controle , Toxina Diftérica/biossíntese , Toxoide Diftérico/administração & dosagem , Monitoramento Epidemiológico , Humanos , Extremidade Inferior/microbiologia , Extremidade Inferior/patologia , Vacinação em Massa , Mutação , Faringe/microbiologia , Faringe/patologia , Pele/microbiologia , Pele/patologia
6.
Diagn Microbiol Infect Dis ; 87(2): 95-99, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27863950

RESUMO

In this study we evaluated the performance of the SHIGA TOXIN QUIK CHEK (Techlab®, Blacksburg, VA) and the ImmunoCard STAT! Enterohaemorrhagic E. coli (EHEC) (Meridian BioScience, Cincinnati, OH, USA) assays as methods for qualitatively detecting the presence of Shiga toxin in human fecal specimens. A multiplex PCR for the detection of stx1 and stx2 was used as the standard for comparison. The SHIGA TOXIN QUIK CHEK detected all known Shiga toxin subtypes with the exception of Stx2f, while the ImmunoCard STAT! EHEC was unable to identify four of the seven Stx2 subtypes, including Stx2b and Stx2d. When compared to multiplex PCR based on Shiga toxin gene presence alone both assays demonstrated 100% specificity, and gave sensitivity values of 50.0% and 41.2% respectively. Correlation between each assay and the multiplex PCR was calculated by the use of kappa, with both assays exhibiting a moderate level of agreement.


Assuntos
Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Fezes/química , Imunoensaio/métodos , Programas de Rastreamento/métodos , Toxina Shiga/análise , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/metabolismo , Humanos , Reação em Cadeia da Polimerase Multiplex , Sensibilidade e Especificidade , Toxina Shiga/genética
7.
PLoS One ; 9(1): e86203, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24475087

RESUMO

Salmonella isolates harbour a range of resident prophages which can influence their virulence and ability to compete and survive in their environment. Phage gene profiling of a range of phage types of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) indicates a significant level of correlation of phage gene profile with phage type as well as correlation with genotypes determined by a combination of multi-locus variable-number tandem repeat (VNTR) typing and clustered regularly interspaced short palindromic repeats (CRISPR) typing. Variation in phage gene profiles appears to be partly linked to differences in composition of variants of known prophages. We therefore conducted a study of the distribution of variants of ST64B and Gifsy-1 prophages and coincidently the presence of Gifsy-3 prophage in a range of S. Typhimurium phage types and genotypes. We have discovered two variants of the DT104 variant of ST64B and at least two new variants of Gifsy-1 as well as variants of related phage genes. While there is definite correlation between phage type and the prophage profile based on ST64B and Gifsy-1 variants we find stronger correlation between the VNTR/CRISPR genotype and prophage profile. Further differentiation of some genotypes is obtained by addition of the distribution of Gifsy-3 and a sequence variant of the substituted SB26 gene from the DT104 variant of ST64B. To explain the correlation between genotype and prophage profile we propose that suites of resident prophages promote clonality possibly through superinfection exclusion systems.


Assuntos
Prófagos/genética , Salmonella typhimurium/genética , Salmonella typhimurium/virologia , DNA Bacteriano/genética , Variação Genética , Tipagem de Sequências Multilocus , Salmonella typhimurium/classificação
8.
Diagn Microbiol Infect Dis ; 77(4): 312-5, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24139878

RESUMO

Campylobacter jejuni is a frequent bacterial pathogen causing gastroenteritis worldwide. We report here a mathematically optimized combination of 10 loci selected from 2 previously published binary typing panels. The optimized combination offers advantages of higher differentiation capability, simplicity, cost-effectiveness, and portability for routine surveillance and outbreak investigations of C. jejuni.


Assuntos
Campylobacter jejuni/classificação , Campylobacter jejuni/genética , Tipagem Molecular/métodos , Animais , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/isolamento & purificação , Genes Bacterianos , Humanos , Tipagem Molecular/normas , Reação em Cadeia da Polimerase Multiplex , Locos de Características Quantitativas
9.
J Microbiol Methods ; 88(1): 19-27, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22015815

RESUMO

In this study we developed a preliminary proof of concept of method for Salmonella typhimurium subtyping using multiplex PCR-based phage locus typing and a multiplex Luminex DNA suspension array for product detection. Thirty markers were selected from prophages ST64B, ST64T, ST104, P22, Gifsy-1, sopEΦ and mostly phage-related AFLP fragments, and organised into two multiplex PCRs of 15 markers each. A two-group DNA suspension array was developed using a combination of flow cytometry and Luminex xMAP® technology. To assess its subtyping capability the method was applied to 438 non-epidemiological related S. typhimurium isolates of 56 phage types. Eighty-one profiles were generated. Isolates were divided into sixteen main prophage marker profile types. There was a strong tendency for isolates with the same phage type to have the same or closely related profiles and for groups of phage types to share the same profile. The discriminatory power of this method expressed as the Simpson's Index of Diversity (D) was 0.954. A panel of 12 selected markers achieved almost the same D value (0.952) as the 30 markers. This new method provides an alternative typing scheme for S. typhimurium epidemiological investigations. The developed array is in a high-throughput format which could easily be semi-automated, making the test fast and economical.


Assuntos
Tipagem de Bacteriófagos/métodos , DNA Bacteriano/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Prófagos/genética , Fagos de Salmonella/genética , Salmonella typhimurium/isolamento & purificação , Proteínas Virais/genética , Técnicas de Tipagem Bacteriana , Dados de Sequência Molecular , Prófagos/fisiologia , Fagos de Salmonella/fisiologia , Salmonella typhimurium/classificação , Salmonella typhimurium/genética , Salmonella typhimurium/virologia
10.
Virology ; 365(1): 187-97, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17462691

RESUMO

Here, we first wished to establish for mouse primary keratinocytes (KCs) the Ca(2+) concentrations that were associated with KC differentiation in vitro. Using the range of Ca(2+) concentrations (0-6 mM) to differentiate primary KCs in culture to varying extents for 2 days, we then examined how KC differentiation impacted on expression of papillomavirus (PV) native (Nat) and codon modified (Mod) L1 genes. L1 mRNAs transcribed from either Nat or Mod L1 genes were present in similar amounts in KCs exposed to six Ca(2+) concentrations. However, expression of the L1 proteins from two Mod L1 genes were down-regulated, with no L1 signal detected in KCs exposed to 6 mM Ca(2+). In contrast, L1 proteins expressed from the two Nat L1 genes were not detectable in KCs without Ca(2+), but dramatically up-regulated as the KC cultures exposed to Ca(2+) from 0.5 to 2 mM, then down-regulated in KCs exposed to Ca(2+) from 4 to 6 mM. The different regulatory roles of the Ca(2+) in L1 protein expression from Nat and Mod L1 genes in cultured KCs were confirmed by TGF-beta1 experiments. We observed that aminoacyl-tRNAs (aa-tRNAs) from the 2 mM Ca(2+)-treated KCs only significantly enhanced the Nat L1 mRNAs translation in vitro, suggesting that aa-tRNAs play a differentially regulatory role in translations of the PV Nat and Mod L1 mRNAs. Importantly, the Ca(2+) experimental model provides evidence that mouse primary KCs could be transiently infected by BPV1 virus to express L1 mRNA and protein, which is very useful for future HPV virus infection study.


Assuntos
Cálcio/metabolismo , Proteínas do Capsídeo/metabolismo , Regulação Viral da Expressão Gênica , Queratinócitos/virologia , Papillomaviridae/fisiologia , Animais , Cálcio/farmacologia , Proteínas do Capsídeo/genética , Diferenciação Celular , Células Cultivadas , Códon , Queratinócitos/citologia , Camundongos , Papillomaviridae/genética , RNA Mensageiro/metabolismo
11.
Mol Cell Biol ; 25(19): 8643-55, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16166644

RESUMO

By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism.


Assuntos
Capsídeo/química , Códon , Regulação Viral da Expressão Gênica , Queratinócitos/citologia , Queratinócitos/virologia , Vírus/metabolismo , Animais , Biolística , Northern Blotting , Western Blotting , Diferenciação Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , DNA/metabolismo , Dactinomicina/farmacologia , Técnicas In Vitro , Queratina-14 , Queratinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Hibridização de Ácido Nucleico , Papillomaviridae/genética , Plasmídeos/metabolismo , Biossíntese de Proteínas , Precursores de Proteínas/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção
12.
J Biol Chem ; 280(4): 2896-903, 2005 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-15494419

RESUMO

The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.


Assuntos
RNA Helicases/química , RNA Helicases/genética , Serina Endopeptidases/química , Serina Endopeptidases/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Vírus do Nilo Ocidental/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cinética , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Eletricidade Estática , Especificidade por Substrato
13.
J Biol Chem ; 279(47): 48535-42, 2004 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-15322074

RESUMO

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G4SG4) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I <0.1 m, 30% glycerol, 1 mm CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR-H, IC50 approximately 1 microm). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.


Assuntos
RNA Helicases/química , Proteínas Recombinantes/química , Serina Endopeptidases/química , Proteínas não Estruturais Virais/química , Vírus do Nilo Ocidental/enzimologia , Sequência de Aminoácidos , Antivirais/farmacologia , Sítios de Ligação , Catálise , Citoplasma/metabolismo , Vírus da Dengue/enzimologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Glicerol/química , Concentração de Íons de Hidrogênio , Hidrólise , Concentração Inibidora 50 , Cinética , Ligantes , Modelos Químicos , Modelos Genéticos , Dados de Sequência Molecular , Peptídeos/química , Conformação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato
14.
Nucleic Acids Res ; 32(15): 4448-61, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15319446

RESUMO

Exogenous transfer RNAs (tRNAs) favor translation of bovine papillomavirus 1 wild-type (wt) L1 mRNA in in vitro translation systems (Zhou et al. 1999, J. Virol., 73, 4972-4982). We, therefore, investigated whether papillomavirus (PV) wt L1 protein expression could be enhanced in eukaryotic cells following exogenous tRNA supplementation. Both Chinese hamster ovary (CHO) and Cos1 cells, transfected with PV1 wt L1 genes, effectively transcribed the genes but did not translate them. However, L1 protein translation was demonstrated following co-transfection with the L1 gene and a gene expressing tRNA(Ser)(CGA). Cell lines, stably transfected with a bovine papillomavirus 1 (BPV1) wt L1 expression construct, produced L1 protein after the transfection of the tRNA(Ser)(CGA) gene, but not following the transfection with basal vectors, suggesting that tRNA(Ser)(CGA) gene enhanced wt L1 translation as a result of endogenous tRNA alterations and phosphorylation of translation initiation factors elF4E and elF2alpha in the tRNA(Ser)(CGA) transfected L1 cell lines. The tRNA(Ser)(CGA) gene expression significantly reduced translation of L1 proteins expressed from codon-modified (HB) PV L1 genes utilizing mammalian preferred codons, but had variable effects on translation of green fluorescent proteins (GFPs) expressed from six serine GFP variants. The changes of tRNA pools appear to match the codon composition of PV wt and HB L1 genes and serine GFP variants to regulate translation of their mRNAs. These findings demonstrate for the first time in eukaryotic cells that translation of the target genes can be differentially influenced by the provision of a single tRNA expression construct.


Assuntos
Proteínas do Capsídeo/genética , Regulação Viral da Expressão Gênica , Biossíntese de Proteínas , RNA de Transferência de Serina/genética , Animais , Papillomavirus Bovino 1/genética , Células CHO , Células COS , Proteínas do Capsídeo/metabolismo , Chlorocebus aethiops , Códon , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Fosforilação , RNA de Transferência/metabolismo , RNA de Transferência de Serina/metabolismo , Transfecção
15.
Protein Expr Purif ; 25(2): 241-7, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12135556

RESUMO

An active form of the Dengue virus protease NS3 (CF40.Gly.NS3pro) was expressed in Escherichia coli. This construct consists of a critical 40 amino acid cofactor domain from NS2B fused to the N-terminal 184 amino acid protease domain of NS3 via a flexible, covalent linker (Gly(4)SerGly(4)). The recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the N-terminus for ease of purification using metal affinity chromatography. However, the presence of lower molecular weight impurities after affinity chromatography indicated the need for additional purification steps. The consistent appearance of these impurities suggested that they may be the products of proteolysis and/or auto-proteolysis. The latter possibility was subsequently excluded by the observation of the same impurities in a purified, catalytically inactive form of the recombinant protease (CF40.Gly.NS3pro.SA). Further analysis indicated that these impurities may represent premature translation termination products. Regardless of their origin, they were shown to form various sized aggregates with full-length CF40.Gly.NS3pro that can be separated by size exclusion chromatography, yielding fractions of active protease of sufficient purity for crystallisation trials. The ultimate goal of these studies is to obtain a crystal structure of a catalytically active form of the Dengue virus NS3 protease for structure-based drug design.


Assuntos
Vírus da Dengue/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Catálise , Cromatografia em Gel , Vírus da Dengue/genética , Complexos Multienzimáticos , RNA Helicases , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Solubilidade , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação
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