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A recombinant hybrid protein as antigen for an anti-blood stage malaria vaccine: a study on the conservation of a protective component

Knapp,Bernhard; Nau,Uwe; Scherf,Artur
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/1992 EN
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45.91%
Recently we have shown that two hybrid proteins expressed in Escherichia coli confer protective immunity to Aotus monkeys against an experimental Plasmodium falciparum infection (Knapp et al., 1992). Both hybrid proteins carry a sequence containing amino acids 631 to 764 of the serine stretch protein SERP (Knapp et al., 1989b). We have studied the diversity of this SERP region in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from different endemic areas of Brazil and West Africa. The SERP region encoding amino acids 630 to 781 was amplified by polymerase chain reaction (PCR) and sequenced. Only conserved amino acid substitutions in maximally two positions of the analyzed SERP fragment could be detected which supports the suitability of this SERP region as a component of anti-blood stage malaria vaccine.

Hybrid Protein between Ribosomal Protein S16 and RimM of Escherichia coli Retains the Ribosome Maturation Function of Both Proteins

Lövgren, J. Mattias; Wikström, P. Mikael
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2001 EN
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46.03%
The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes and is important for efficient maturation of the 30S subunits. A mutant lacking RimM shows a sevenfold-reduced growth rate and a reduced translational efficiency. Here we show that a double alanine-for-tyrosine substitution in RimM prevents it from associating with the 30S subunits and reduces the growth rate of E. coli approximately threefold. Several faster-growing derivatives of the rimM amino acid substitution mutant were found that contain suppressor mutations which increased the amount of the RimM protein by two different mechanisms. Most of the suppressor mutations destabilized a secondary structure in the rimM mRNA, which previously was shown to decrease the synthesis of RimM by preventing the access of the ribosomes to the translation initiation region on the rimM mRNA. Three other independently isolated suppressor mutations created a fusion between rpsP, encoding the ribosomal protein S16, and rimM on the chromosome as a result of mutations in the rpsP stop codon preceding rimM. A severalfold-higher amount of the produced hybrid S16-RimM protein in the suppressor strains than of the native-sized RimM in the original substitution mutant seems to explain the suppression. The S16-RimM protein but not any native-size ribosomal protein S16 was found both in free 30S ribosomal subunits and in translationally active 70S ribosomes of the suppressor strains. This suggests that the hybrid protein can substitute for S16...

Humoral and Cellular Immune Responses in Mice Immunized with Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Producing a Pertussis Toxin-Tetanus Toxin Hybrid Protein

Abomoelak, B.; Huygen, K.; Kremer, L.; Turneer, M.; Locht, C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/1999 EN
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45.87%
The development of combined vaccines constitutes one of the priorities in modern vaccine research. One of the most successful combined vaccines in use is the diphtheria-pertussis-tetanus vaccine. However, concerns about the safety of the pertussis arm have led to decreased acceptance of the vaccine but also to the development of new, safer, and effective acellular vaccines against pertussis. Unfortunately, the production cost of these new vaccines is significantly higher than that of previous vaccines. Here, we explore the potential of live recombinant Mycobacterium bovis BCG producing the hybrid protein S1-TTC, which contains the S1 subunit of pertussis toxin fused to fragment C of tetanus toxin, as an alternative to the acellular vaccines. S1-TTC was produced in two different expression systems. In the first system its production was under the control of the 85A antigen promoter and signal peptide, and in the second system it was under the control of the hsp60 promoter. Although expression of the hybrid antigen was obtained in both cases, only the second expression system yielded a recombinant BCG strain able to induce both a specific humoral immune response and a specific cellular immune response. The antibodies generated were directed against the TTC part and neutralized toxin activity in an in vivo challenge model...

Diverse effects of the MalE-LacZ hybrid protein on Escherichia coli cell physiology.

Ito, K; Akiyama, Y; Yura, T; Shiba, K
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1986 EN
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46.13%
The hybrid protein between the periplasmic maltose-binding protein and the cytoplasmic beta-galactosidase (the MalE-LacZ hybrid protein) was previously shown to block the export of envelope proteins when synthesized in large amounts. Now we show that the hybrid protein exerts another major effect on the cell, that is, induction of the heat shock proteins. This latter effect was dependent on the htpR gene product but independent of the function of the signal sequence on the hybrid protein. On the other hand, the previously reported induction of the SecA protein by the hybrid protein was independent of htpR and may be caused by the reduced protein export ability of the cell. The functional htpR gene is essential for viability of the cell in which the basal level of the hybrid protein is synthesized, whereas in the absence of the hybrid protein htpR is dispensable at low temperature. These results indicate that the hybrid protein somehow generates a signal or stress that is similar to what the cell experiences at elevated temperatures.

Use of a hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin and part of the 83-kDa protein as antigen for serodiagnosis of Lyme disease.

Rasiah, C; Rauer, S; Gassmann, G S; Vogt, A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1994 EN
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46.06%
A hybrid protein consisting of the variable region of the Borrelia burgdorferi flagellin (an 18-kDa fragment) and a 59-kDa fragment (lacking the N-terminal part) of the 83-kDa protein has been constructed by genetic engineering. It was expressed as a nonfusion protein of an apparent molecular weight of 77,000 in Escherichia coli. The suitability of this new antigen for the diagnosis of Lyme disease was tested by immunoblotting; for comparison, the recombinant variable region of the flagellin, the 18-kDa fragment (p18), and the whole recombinant 83-kDa protein (p83), both expressed in E. coli, were used. A total of 120 serum samples from various stages of Lyme disease, which were positive in two serological assays, a passive hemagglutination assay and an indirect immunofluorescence assay, were tested. By indirect immunofluorescence, 74 samples were positive for immunoglobulin G (IgG) antibodies and 72 were positive for IgM antibodies. Of these serum samples, 69 of 74 (93%) contained IgG antibodies against p18 and/or p83, and IgG antibodies were detected by the hybrid protein in 67 (90%) samples. IgM antibodies against p18 and/or p83 were detected in 60 of 72 (83%) serum samples, and 57 (79%) serum samples were reactive with the hybrid protein. Twenty serum samples of patients with a history of syphilis and 40 serum samples...

Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria.

Douglas, M G; Geller, B L; Emr, S D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1984 EN
Relevância na Pesquisa
46.06%
The gene coding for the yeast mitochondrial F1-ATPase beta subunit (ATP2) has been fused to the Escherichia coli lacZ gene. The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase beta subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), at its carboxyl terminus. The beta-subunit-beta-galactosidase hybrid protein is expressed in both E. coli and yeast. In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested. Yeast cells carrying the ATP2-lacZ gene fusion on plasmid p beta Z1 are unable to grow on a nonfermentable carbon source. Upon loss of the p beta Z1 plasmid, growth of the cured host strain on the nonfermentable substrate is restored. In the presence of the beta-subunit-beta-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product. The results indicate that it is the presence of the beta-subunit-beta-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth. These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery.

Protein kinase activity associated with the D2 hybrid protein related to simian virus 40 T antigen: Some characteristics of the reaction products

Baumann, Ernst A.; Hand, Roger
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1979 EN
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46.03%
Protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) has been found associated with the D2 hybrid protein, a highly purified protein of 107,000 daltons specified by the adenovirus-simian virus 40 (SV40) hybrid Ad2+D2, which has many properties associated with authentic SV40 T antigen [Tjian, R. & Robbins, A. (1979) Proc. Natl. Acad. Sci. USA 76, 610-614]. We have now examined some of the biochemical characteristics of the reaction products. Acceptors for the terminal phosphoryl group of [γ-32P]ATP are the purified protein itself and at least four proteins extracted from nuclei of uninfected cells. Purified histones do not serve as substrate for the enzyme. Phosphorylation is markedly reduced by heating the D2 hybrid protein to 50°C for 30 min. The products of phosphorylation are stable to treatment with ethanol/ether, DNase, and RNase, but completely degraded by digestion with Pronase, demonstrating their protein nature. The phosphate bonds are liable to hot alkali and sensitive to digestion with alkaline phosphatase but stable to treatment with hot acid or hydroxylamine. These results provide evidence that 32P is incorporated into O-phosphoserine or O-phosphothreonine residues of acceptor proteins, indicating that the enzymatic activity is characteristic for protein kinase...

Expression of a Functional Secreted YopN-TyeA Hybrid Protein in Yersinia pestis Is the Result of a +1 Translational Frameshift Event

Ferracci, Franco; Day, James B.; Ezelle, Heather J.; Plano, Gregory V.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2004 EN
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45.93%
YopN is a secreted protein that prior to secretion directly interacts with the cytosolic SycN/YscB chaperone complex and TyeA. This study identifies a secreted YopN-TyeA hybrid protein that is expressed by Yersinia pestis, but not by Yersinia enterocolitica. DNA sequence analysis and site-directed mutagenesis studies demonstrate that the hybrid protein is the result of a +1 translational frameshift event.

HYPROSP: a hybrid protein secondary structure prediction algorithm—a knowledge-based approach

Wu, Kuen-Pin; Lin, Hsin-Nan; Chang, Jia-Ming; Sung, Ting-Yi; Hsu, Wen-Lian
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
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45.95%
We develop a knowledge-based approach (called PROSP) for protein secondary structure prediction. The knowledge base contains small peptide fragments together with their secondary structural information. A quantitative measure M, called match rate, is defined to measure the amount of structural information that a target protein can extract from the knowledge base. Our experimental results show that proteins with a higher match rate will likely be predicted more accurately based on PROSP. That is, there is roughly a monotone correlation between the prediction accuracy and the amount of structure matching with the knowledge base. To fully utilize the strength of our knowledge base, a hybrid prediction method is proposed as follows: if the match rate of a target protein is at least 80%, we use the extracted information to make the prediction; otherwise, we adopt a popular machine-learning approach. This comprises our hybrid protein structure prediction (HYPROSP) approach. We use the DSSP and EVA data as our datasets and PSIPRED as our underlying machine-learning algorithm. For target proteins with match rate at least 80%, the average Q3 of PROSP is 3.96 and 7.2 better than that of PSIPRED on DSSP and EVA data, respectively.

Immunization of Saimiri sciureus Monkeys with a Recombinant Hybrid Protein Derived from the Plasmodium falciparum Antigen Glutamate-Rich Protein and Merozoite Surface Protein 3 Can Induce Partial Protection with Freund and Montanide ISA720 Adjuvants

Carvalho, Leonardo J. M.; Alves, Francisco A.; Bianco, Cesare; Oliveira, Salma G.; Zanini, Graziela M.; Soe, Soe; Druilhe, Pierre; Theisen, Michael; Muniz, José A. P. C.; Daniel-Ribeiro, Cláudio T.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2005 EN
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45.97%
The immunogenicity and efficacy of a hybrid recombinant protein derived from the N-terminal end of the glutamate-rich protein (GLURP) and the C-terminal portion of the merozoite surface protein 3 (MSP3) of Plasmodium falciparum was evaluated in Saimiri sciureus monkeys. The GLURP/MSP3 hybrid protein, expressed in Lactococcus lactis, was administered in association with alum, Montanide ISA720, or complete or incomplete Freund adjuvant (CFA/IFA) in groups of five animals each. The three formulations were shown to be immunogenic, but the one with alum was shown to be weak compared to the other two, particularly CFA/IFA, which provided very high antibody titers (enzyme-linked immunosorbent assay titers of >3,000,000 and immunofluorescence antibody test titers of 6,400). After a challenge infection with P. falciparum FUP strain, all five monkeys from the GLURP/MSP3-alum group showed a rapid increase in parasitemia, reaching 10% and were treated early. The two monkeys with the highest antibody titers in group GLURP/MSP3-Montanide ISA720 had a delay in the course of parasitemia and were treated late due to a low hematocrit. In the GLURP/MSP3-CFA/IFA group, parasitemia remained below this threshold in four of the five animals and, after it reached a peak...

Ethylnitrosourea-induced base pair substitution affects splicing of the mouse gammaE-crystallin encoding gene leading to the expression of a hybrid protein and to a cataract.

Graw, Jochen; Neuhäuser-Klaus, Angelika; Löster, Jana; Klopp, Norman; Favor, Jack
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/2002 EN
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45.87%
A novel ENU-induced mutation in the mouse leading to a nuclear and cortical opacity of the eye lens (ENU418) was mapped to proximal chromosome 1 by a genome-wide mapping approach. It suggests that the cluster of gamma-crystallin encoding genes (Cryg) and the betaA2-crystallin encoding gene Cryba2 are excellent candidate genes. An A --> G exchange in the middle of intron 1 of the Cryge gene was found as the only alteration cosegregating with the cataractous phenotype. The mutation was confirmed by the presence of a novel restriction site for ApaI in the corresponding genomic DNA fragment. The mutation represses splicing of intron 1; the additional 92 bp in the corresponding cDNA leads to a frameshift and the expression of a novel hybrid protein containing 3 amino acids of the gammaE-crystallin at the N terminus, but 153 novel amino acids. The Cryge(ENU418) protein has a calculated molecular mass of approximately 15.6 kD and an alkaline isoelectric point (pH 10.1) and is predicted to have two hydrophobic domains. Western blot analysis using a polyclonal antibody against the hydrophilic C-terminal part of the Cryge(ENU418)-specific protein demonstrated its stable expression in the cataractous lenses; it was not found in the wild types. Histological analysis of the cataractous lenses indicated that the expression of the new protein disrupts the cellular structure of the eye lens.

Interaction with Polyglutamine-expanded Huntingtin Alters Cellular Distribution and RNA Processing of Huntingtin Yeast Two-hybrid Protein A (HYPA)*

Jiang, Ya-Jun; Che, Mei-Xia; Yuan, Jin-Qiao; Xie, Yuan-Yuan; Yan, Xian-Zhong; Hu, Hong-Yu
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
45.86%
Huntington disease (HD) is an autosomal inherited disorder that causes the deterioration of brain cells. The polyglutamine (polyQ) expansion of huntingtin (Htt) is implicated in the pathogenesis of HD via interaction with an RNA splicing factor, Htt yeast two-hybrid protein A/forming-binding protein 11 (HYPA/FBP11). Besides the pathogenic polyQ expansion, Htt also contains a proline-rich region (PRR) located exactly in the C terminus to the polyQ tract. However, how the polyQ expansion influences the PRR-mediated protein interaction and how this abnormal interaction leads to the biological consequence remain elusive. Our NMR structural analysis indicates that the PRR motif of Htt cooperatively interacts with the tandem WW domains of HYPA through domain chaperoning effect of WW1 on WW2. The polyQ-expanded Htt sequesters HYPA to the cytosolic location and then significantly reduces the efficiency of pre-mRNA splicing. We propose that the toxic gain-of-function of the polyQ-expanded Htt that causes dysfunction of cellular RNA processing contributes to the pathogenesis of HD.

A polyvalent hybrid protein elicits antibodies against the diverse allelic types of block 2 in Plasmodium falciparum merozoite surface protein 1

Tetteh, Kevin K.A.; Conway, David J.
Fonte: Elsevier Science Publicador: Elsevier Science
Tipo: Artigo de Revista Científica
Publicado em 13/10/2011 EN
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46.04%
Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice. The full hybrid protein induced high titre antibodies against each of the major block 2 allelic types expressed as separate recombinant proteins and against a wide range of allelic types naturally expressed by a panel of diverse P. falciparum isolates, while the sub-component modules had partial antigenic coverage as expected. This encourages further development and evaluation of the full MSP1 block 2 polyvalent hybrid protein as a candidate blood-stage component of a malaria vaccine.

Comparative Molecular Modeling Study of Arabidopsis NADPH-Dependent Thioredoxin Reductase and Its Hybrid Protein

Lee, Yuno; Kim, Songmi; Lazar, Prettina; Moon, Jeong Chan; Hwang, Swan; Thangapandian, Sundarapandian; Shon, Youngsik; Lee, Kyun Oh; Lee, Sang Yeol; Lee, Keun Woo
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 27/09/2012 EN
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45.91%
2-Cys peroxiredoxins (Prxs) play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC) was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C) of thioredoxin reductase (TrxR) in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx) domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D)), which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD) simulations on AtNTRC and AtNTRA-(Trx-D) proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD) for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D) cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D) because of following reasons: i) unstable and unfavorable interaction of the linker region...

Complement factor H–related hybrid protein deregulates complement in dense deposit disease

Chen, Qian; Wiesener, Michael; Eberhardt, Hannes U.; Hartmann, Andrea; Uzonyi, Barbara; Kirschfink, Michael; Amann, Kerstin; Buettner, Maike; Goodship, Tim; Hugo, Christian; Skerka, Christine; Zipfel, Peter F.
Fonte: American Society for Clinical Investigation Publicador: American Society for Clinical Investigation
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
45.98%
The renal disorder C3 glomerulopathy with dense deposit disease (C3G-DDD) pattern results from complement dysfunction and primarily affects children and young adults. There is no effective treatment, and patients often progress to end-stage renal failure. A small fraction of C3G-DDD cases linked to factor H or C3 gene mutations as well as autoantibodies have been reported. Here, we examined an index family with 2 patients with C3G-DDD and identified a chromosomal deletion in the complement factor H–related (CFHR) gene cluster. This deletion resulted in expression of a hybrid CFHR2-CFHR5 plasma protein. The recombinant hybrid protein stabilized the C3 convertase and reduced factor H–mediated convertase decay. One patient was refractory to plasma replacement and exchange therapy, as evidenced by the hybrid protein quickly returning to pretreatment plasma levels. Subsequently, complement inhibitors were tested on serum from the patient for their ability to block activity of CFHR2-CFHR5. Soluble CR1 restored defective C3 convertase regulation; however, neither eculizumab nor tagged compstatin had any effect. Our findings provide insight into the importance of CFHR proteins for C3 convertase regulation and identify a genetic variation in the CFHR gene cluster that promotes C3G-DDD. Monitoring copy number and sequence variations in the CFHR gene cluster in C3G-DDD and kidney patients with C3G-DDD variations will help guide treatment strategies.

Two-hybrid analysis of the interaction between the fission yeast proteins Sal3 and Cdc25

Perinpanayagam, Gajendran
Fonte: Quens University Publicador: Quens University
Tipo: Tese de Doutorado Formato: 1442268 bytes; application/pdf
EN
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45.95%
Cdc25 is a mitosis triggering phosphatase in Schizosaccharomyces pombe, and is transported in to the nucleus during G2 phase by the importin-β protein Sal3. Cdc25 triggers mitosis and cell division by dephosphorylating tyrosine 15 of Cdc2. In sal3 mutants, Cdc25 is not transported into the nucleus and the cells halt in G2. The purpose of this study is to use a two-hybrid system to determine the nature of the relationship between Sal3 and Cdc25. Previous research has failed to detect any interaction between the two proteins, but specific modifications were made to the two-hybrid system in this study including the separation of Sal3 into its two binding domains, the addition of fluorescent tags to the fusion protein, and the reversal of plasmids in the fusion proteins. Unique PCR primers were successfully designed, based on a multiple alignment of Sal3 and its homologues, to separate Sal3 into its two domains.

Expression of an equine herpesvirus 1 ICP22/ICP27 hybrid protein encoded by defective interfering particles associated with persistent infection.

Chen, M; Harty, R N; Zhao, Y; Holden, V R; O'Callaghan, D J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1996 EN
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46.07%
Defective interfering (DI) particles of equine herpesvirus type 1 (EHV-1) are capable of mediating persistent infection (S. A. Dauenhauer, R. A. Robinson, and D. J. O'Callaghan, J. Gen. Virol. 60:1-14, 1982; R. A. Robinson, R. B. Vance, and D. J. O'Callaghan, J. Virol. 36:204-219, 1980). Sequence analysis of cloned DI particle DNA revealed that portions of two regulatory genes, ICP22 (IR4) and ICP27 (UL3), are linked in frame to form a unique hybrid open reading frame (ORF). This hybrid ORF, designated as the IR4/UL3 gene, encodes the amino-terminal 196 amino acids of the IR4 protein (ICP22 homolog) and the carboxy-terminal 68 amino acids of the UL3 protein (ICP27 homolog). Portions of DNA sequences encoding these two regulatory proteins, separated by more than 115 kbp in the standard virus genome, were linked presumably by a homologous recombination event between two identical 8-bp sequences. Reverse transcriptase-PCR and S1 nuclease analyses revealed that this unique ORF is transcribed by utilizing the transcription initiation site of ICP22 and the polyadenylation signal of ICP27 in DI particle-enriched infection. Immunoprecipitation and Western blot (immunoblot) analyses with antisera to the ICP22 and ICP27 proteins demonstrated that a 31-kDa hybrid protein was synthesized in the DI particle-enriched infection but not in standard virus infection. This 31-kDa hybrid protein was expressed at the same time as the ICP22 protein in DI particle-enriched infection and migrated at the same location on polyacrylamide gel electrophoresis as the protein expressed from a cloned IR4/UL3 expression vector. These observations suggested that the unique IR4/UL3 hybrid gene is expressed from the DI particle genome and may play a role in DI particle-mediated persistent infection.

Intracellular Targeting and Import of an F1-ATPase β-subunit-β-galactosidase Hybrid Protein into Yeast Mitochondria

Douglas, Michael G.; Geller, Bruce L.; Emr, Scott D.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Article; PeerReviewed Formato: application/pdf
Publicado em 01/07/1984
Relevância na Pesquisa
46.06%
The gene coding for the yeast mitochondrial F1-ATPase β subunit (ATP2) has been fused to the Escherichia coli lacZ gene. The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase β subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23), at its carboxyl terminus. The β-subunit-β -galactosidase hybrid protein is expressed in both E. coli and yeast. In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested. Yeast cells carrying the ATP2-lacZ gene fusion on plasmid pβ Z1 are unable to grow on a nonfermentable carbon source. Upon loss of the pβ Z1 plasmid, growth of the cured host strain on the nonfermentable substrate is restored. In the presence of the β-subunit-β-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product. The results indicate that it is the presence of the β-subunit-β-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth. These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery.

The Chlamydia Trachomatis Protein Interaction Network: Insights into the Unique Composition of the Type Three Secretion System

Spaeth, Kris Edmund
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação Formato: 2922617 bytes; application/pdf
Publicado em 19/11/2008 EN_US
Relevância na Pesquisa
45.87%

The Gram-negative bacteria Chlamydia trachomatis is a common sexually transmitted pathogen that can cause severe sequelae including cause pelvic inflammatory disease and sterility. This obligate intracellular pathogen effectively manipulates host cellular functions by secreting virulence factors across its membrane bound vacuole. Identifying these virulence components and how they help in establishing an environment conducive for bacterial growth is central to understanding chlamydial pathogenesis. This is experimentally challenging due to a lack of tools to perform molecular genetic studies. In the absence of genetic tools, we developed a yeast model system to identify and characterize chlamydial proteins involved in virulence mechanisms. In this study we describe the identification of twenty-eight proteins potentially involved in modulating host cellular functions and the secretion of virulence factors into the host. Since the delivery of virulence proteins by a type three secretion (T3S) system is a critical step for Chlamydia, we identified the proteins that interacted with the T3S apparatus by yeast two-hybrid analysis. We discovered several novel interactions between and determined that the C. trachomatis T3S apparatus displayed a similar architecture to that of other T3S systems. Furthermore with these approaches we identified networks of proteins that interacted with the secretion apparatus including a novel secretion chaperone protein. We characterized Ct260/Mcsc one of the putative secretion and demonstrated that it represents a novel class 1B secretion chaperone protein. Unlike other known chaperones...

The Interdomain Region of Dengue NS5 Protein That Binds to the Viral Helicase NS3 Contains Independently Functional Importin beta1 and importin alpha/beta -Recognised Nuclear Localization Signals

Brooks, Andrew J; Johansson, Magnus; John, Anna; Xu, Yibin; Jans, David A; Vasudevan, Subhash
Fonte: American Society for Biochemistry and Molecular Biology Inc Publicador: American Society for Biochemistry and Molecular Biology Inc
Tipo: Artigo de Revista Científica
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45.86%
Dengue virus NS5 protein is a multifunctional RNA-dependent RNA polymerase that is essential for virus replication. We have shown previously that the 37-amino acid interdomain spacer sequence (residues369X2KKX14KKKX11 RKX3405) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, β-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369-389 or residues 386-405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369-389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin α/β heterodimeric nuclear import receptor. We also show that the importin β-binding site (residues 320-368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin β, and able to target β-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin β in the infectious cycle.