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2. Rapid RNase inhibitor production to enable low-cost, on-demand cell-free protein synthesis biosensor use in human body fluids.
- Author
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Soltani M, Hunt JP, and Bundy BC
- Subjects
- Animals, Cell-Free System, Humans, Mice, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Biosensing Techniques, Body Fluids metabolism, Escherichia coli chemistry, Proteins chemistry
- Abstract
Human body fluids contain biomarkers which are used extensively for prognostication, diagnosis, monitoring, and evaluation of different treatments for a variety of diseases and disorders. The application of biosensors based on cell-free protein synthesis (CFPS) offers numerous advantages including on-demand and at-home use for fast, accurate detection of a variety of biomarkers in human fluids at an affordable price. However, current CFPS-based biosensors use commercial RNase inhibitors to inhibit different RNases present in human fluids and this reagent is approximately 90% of the expense of these biosensors. Here the flexible nature of Escherichia coli-lysate-based CFPS was used for the first time to produce murine RNase Inhibitor (m-RI) and to optimize its soluble and active production by tuning reaction temperature, reaction time, reduced potential, and addition of GroEL/ES folding chaperons. Furthermore, RNase inhibition activity of m-RI with the highest activity and stability was determined against increasing amounts of three human fluids of serum, saliva, and urine (0%-100% v/v) in lyophilized CFPS reactions. To further demonstrate the utility of the CFPS-produced m-RI, a lyophilized saliva-based glutamine biosensor was demonstrated to effectively work with saliva samples. Overall, the use of CFPS-produced m-RI reduces the total reagent costs of CFPS-based biosensors used in human body fluids approximately 90%., (© 2021 Wiley Periodicals LLC.)
- Published
- 2021
- Full Text
- View/download PDF
3. CHROMATOGRAPHIC STUDIES ON AMINOACYL-ACCEPTOR RNA USING PAPER SHEETS OF DIETHYLAMINOETHYL-CELLULOSE.
- Author
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JACOBSON KB and NISHIMURA S
- Subjects
- Carbon Isotopes, Cellulose, Chromatography, Escherichia coli, Leucine, Proteins, RNA, RNA, Bacterial, RNA, Messenger, Research, Sodium Chloride
- Published
- 1964
- Full Text
- View/download PDF
4. Microbial production of butyl butyrate, a flavor and fragrance compound.
- Author
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Noh HJ, Lee SY, and Jang YS
- Subjects
- Clostridium acetobutylicum genetics, Escherichia coli genetics, Lipase genetics, Metabolic Networks and Pathways genetics, Proteins genetics, Butyrates metabolism, Clostridium acetobutylicum metabolism, Escherichia coli metabolism, Lipase metabolism, Metabolic Engineering methods, Proteins metabolism
- Abstract
Butyl butyrate (BB) has been widely used as a flavor and fragrance compound in the beverage, food, perfume, and cosmetic industries. Currently, BB is produced through two-step processes; butanol and butyrate are first produced and are used as precursors for the esterification reactions to yield BB in the next step. Recently, an alternative process to the current process has been developed by using microorganisms for the one-pot BB production. In the one-pot BB process, alcohol acyl transferases (AATs) and lipases play roles in the esterification of butanol together with their co-substrates butyryl-CoA and butyrate, respectively. In this paper, we review the characteristics of two enzymes including AAT and lipase in the esterification reaction. Also, we review the one-pot processes for BB production by employing the wild-type and engineered Clostridium species and the engineered Escherichia coli strains, with the combination of AATs and lipases.
- Published
- 2019
- Full Text
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5. Les famílies de proteïnes Hha/YmoA i H-NS: regulació de l'expressió gènica a 'Escherichia coli' i paper de la conjugació plasmídica
- Author
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Forns Fradera, Núria, Madrid Xufré, Cristina, and Universitat de Barcelona. Departament de Microbiologia
- Subjects
Transcripció genètica ,Escheríchia coli ,Enterobacteriaceae ,Genetic transcription ,Escherichia coli ,Proteins ,Gene expression ,Expressió gènica ,Proteïnes ,Enterobacteriàcies - Abstract
[cat] En bacteris entèrics, les proteïnes de la família Hha/YmoA estan implicades en la regulació de l'expressió gènica depenent factors ambientals. Ha estat descrit que la proteïna Hha d' Escherichia coli interacciona amb la proteïna H-NS formant un complex amb el DNA responsable de la termoregulació de l'expressió de l'operó hly d' E. coli. Altres proteïnes d'aquesta família, com YmoA i YdgT, també interaccionen amb la proteïna H-NS.La proteïna H-NS d' E. coli és una proteïna associada al nucleoide implicada en la regulació global de l'expressió gènica en funció de les condicions ambientals. H-NS es troba àmpliament distribuida entre bacteris Gram-negatius.El primer objectiu va ser determinar si la participació conjunta de les proteïnes Hha i H-NS era extensible a altres gens regulats per H-NS. Escollirerm l'operó bgl, silenciat per la proteïna H-NS. Mitjançant mesures de l'activitat i de la trasncripció de l'operó vam concloure que les proteïnes Hha i YdgT intervenen en el silenciament de l'operó bgl d'Escherichia coli.Es va analitzar la regulació de l'expressió gènica depenent de les proteïnes Hha i H-NS en diferents condicions d'osmolaritat per identificar les proteïnes diferencialment expressades. S'identificaren les proteïnes HdeA, l'expressió de la qual ja havia estat descrita com a regulada per H-NS; i HtrA, una proteasa sotmesa a osmoregulació. El complex Hha-H-NS és responsable de la repressió de l'expressió d'HtrA a baixa osmolaritat.En la seqüència completa del plàsmid R27 s'han identificat dos gens (orf182 i orf164) que codifiquen dues proteïnes homòlogues a Hha i H-NS, respectivament. Ja que la conjugació d'aquest plàsmid està sotmesa a termoregulació, i donat que les proteïnes Hha i H-NS estan implicades en la regulació de l'expressió gènica depenent de paràmetres ambientals, es va voler determinar: · A) el paper d'aquestes proteïnes, d'origen plasmídic i d'origen cromosòmic, en la regulació de la conjugació de R27,· B) i la intervenció de les proteïnes de codificació plasmídica en la fisiologia de la cèl·lula portadora del plàsmid. Es realitzaren estudis de la freqüència de conjugació del plàsmid, estudis de la transcripció dels gens de transferencia mitjançant microxips i RT-PCR, assaigs de retard en gel amb les proteïnes Hha i H-NS i observació de pilis per MET. Es va concloure que les proteïnes Hha i H-NS, tant de codificació plasmídica com cromosòmica, interven en la termoregulació de la conjugació del plàsmid, reprimint l'expressió dels gens de transferencia a elevada temperatura. En estudis de complementació s'observà que les proteïnes codificades als gens orf182 i orf164, compensen alguns fenotips causats per les mutacions dels gens hha i hns. Es va demostrar també la interacció in vitro de la proteïna H-NS plasmídica (ORF164) amb la proteïna Hha cromosòmica.Finalment, es va realitzar una anàlisi trancriptòmica de l'efecte de les mutacions hns i hha ydgT en el patró d'expressió gènica a Escherichia coli, per determinar quins gens estaven afectats en cada fons genètic i establir la relació entre la regulació per H-NS i per Hha/YdgT. La comparació dels patrons d'expressió entre la soca hns i la soca salvatge, han posat de manifest que un 7% dels gens d'E. coli presenten una alteració significativa de la seva expressió, i que un 68% dels casos corresponen a una sobreexpressió en la soca hns. L'analisi de l'expressió diferencial entre la soca hha ydgT i la soca hns demostra que la regulació de la soca hha ydgT segueix majoritàriment el mateix patró d'expressió que la soca salvatge. Es van identificar 22 gens amb expressió diferencial entre la soca hha ydgT i la soca salvatge, dels quals 18 presenten sobreexpressió en un fons genètic hha ydgT, i 12 coincideixen amb els gens regulats per H-NS., [eng] In enteric bacteria, the proteins of Hha/YmoA family play an important role in regulation of gene expression in response to environmental signals. On another part, the nucleoid-associated protein H-NS is a relevant example of a global modulator. H-NS binds to Hha, and other proteins of Hha/YmoA family, to regulate gene expression depending on environmental conditions.The first goal was to determine if Hha and H-NS participate together regulating genes known as regulated by H-NS, like "bgl" operon silenced by H-NS, or other new genes in different osmolarity conditions. We conclude that Hha play a role in silencing "bgl" operon expression in "E. Coli" and that Hha-H-NS complex is responsible for the HtrA repression at low osmolarity.R27 plasmid code two proteins homologous to Hha and H-NS, respectively. Since R27 conjugation is thermoregulated, and given the fact that Hha and H-NS regulate gene expression depending on environmental parameters, we investigated the role of these proteins, in thermoregulation of R27 conjugation. We conclude that Hha and H-NS, of plasmidic and chromosomic origin, downregulate plasmid conjugation at high temperature. As well, we studied the intervention of Hha and H-NS R27 encoded proteins in the physiology of R27 host cells. We observed that these proteins compensate some of the phenotypes caused by hha and hns mutations.Finally, a transcriptomic analysis of the effect of the mutations "hns" and "hha ydgT" in "E. coli"i was carried out to establish the relationship between H-NS and Hha/YdgT regulation. The result showed that 7% of genes were affected by "hns" mutation and that 68% of these cases corresponds to an overexpression in hns strain. The analysis of "hha ydgT" strain demonstrates that its regulation follows mostly the same pattern of expression than the wild type strain, because "hha ydgT" mutations only affected 22 genes.
- Published
- 2006
6. Three-dimensional structure determination of proteins related to human health in their functional context at The Israel Structural Proteomics Center (ISPC). This paper was presented at ICCBM10
- Author
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Shira, Albeck, Yigal, Burstein, Orly, Dym, Yossi, Jacobovitch, Nurit, Levi, Ran, Meged, Yigal, Michael, Yoav, Peleg, Jaime, Prilusky, Gideon, Schreiber, Israel, Silman, Tamar, Unger, and Joel L, Sussman
- Subjects
Inclusion Bodies ,Models, Molecular ,Proteomics ,Internet ,Protein Denaturation ,Protein Folding ,Protein Conformation ,Computational Biology ,Proteins ,DNA ,Pichia ,Recombinant Proteins ,Automation ,Escherichia coli ,Humans ,Electrophoresis, Polyacrylamide Gel ,Cloning, Molecular ,Israel ,Databases, Protein - Abstract
The principal goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context. Emphasis is on the solution of structures of proteins complexed with their natural partner proteins and/or with DNA. To date, the ISPC has solved the structures of 14 proteins, including two protein complexes. It has adopted automated high-throughput (HTP) cloning and expression techniques and is now expressing in Escherichia coli, Pichia pastoris and baculovirus, and in a cell-free E. coli system. Protein expression in E. coli is the primary system of choice in which different parameters are tested in parallel. Much effort is being devoted to development of automated refolding of proteins expressed as inclusion bodies in E. coli. The current procedure utilizes tagged proteins from which the tag can subsequently be removed by TEV protease, thus permitting streamlined purification of a large number of samples. Robotic protein crystallization screens and optimization utilize both the batch method under oil and vapour diffusion. In order to record and organize the data accumulated by the ISPC, a laboratory information-management system (LIMS) has been developed which facilitates data monitoring and analysis. This permits optimization of conditions at all stages of protein production and structure determination. A set of bioinformatics tools, which are implemented in our LIMS, is utilized to analyze each target.
- Published
- 2004
7. Microspheres of mixed proteins.
- Author
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Angel Shimanovich U, Matas D, Michaeli S, Cavaco-Paulo A, and Gedanken A
- Subjects
- Animals, Cattle, Green Fluorescent Proteins metabolism, Humans, Micelles, Microscopy, Fluorescence, Microspheres, Molecular Structure, Particle Size, Protein Binding, Serum Albumin metabolism, Serum Albumin, Bovine metabolism, Surface Properties, Escherichia coli chemistry, Escherichia coli Proteins chemistry, Green Fluorescent Proteins chemistry, Proteins chemistry, Serum Albumin chemistry, Serum Albumin, Bovine chemistry
- Abstract
This paper describes the synthesis of mixed proteinaceous microspheres (MPMs) by the sonochemical method. The current fundamental research follows the research of Suslick and co-workers who have developed a method by which high-intensity ultrasound is used to make aqueous suspensions of proteinaceous microcapsules filled with water-insoluble liquids.1 By using high-intensity ultrasound, we have synthesized microspheres made of a few different proteins. The three proteins used in the current experiments are bovine serum albumin (BSA), green fluorescent protein (GFP), and cyan fluorescent protein-glucose binding protein-yellow fluorescent fused protein (CFP-GBP-YFP). The two synthesized microspheres made of mixed proteins are BSA-GFP and BSA-(CFP-GBP-YFP). This paper presents the characterization of the sonochemically produced microspheres of mixed proteins. It also provides an estimate of the efficiency of the sonochemical process in converting the native proteins to microspheres.
- Published
- 2010
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8. Vascular endothelial cell antigen (VECA), a novel secreted protein: overexpression in Escherichia coli of the hexahistidine- tagged protein and production of polyclonal antibodies.
- Author
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Xiong SQ, Yang HS, Chen YY, Zhao CJ, Yuan Z, Zhao XY, Yan F, Wang BD, Tang MH, Yang JL, and Wei YQ
- Subjects
- Amino Acid Sequence, Antibodies metabolism, Antigens genetics, Antigens isolation & purification, Blotting, Western, Breast Neoplasms immunology, Breast Neoplasms pathology, Cloning, Molecular methods, Escherichia coli metabolism, Female, Humans, Immunohistochemistry, Molecular Sequence Data, Protein Structure, Tertiary, Proteins chemistry, Proteins metabolism, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins immunology, Spectrometry, Mass, Electrospray Ionization, Uterine Neoplasms immunology, Uterine Neoplasms pathology, Antibodies immunology, Antigens biosynthesis, Antigens immunology, Endothelial Cells immunology, Escherichia coli genetics, Proteins immunology
- Abstract
A novel VECA (vascular endothelial cell antigen) was previously identified by using an antibody pool against antigens in HUVECs (human umbilical-vein endothelial cells). VECA has been evolutionarily conserved in vertebrate species ranging from frog and fish to mouse and human. Bioinformatics analysis indicated that VECA was 10.1 kDa in size, with a predicted signal sequence and transmembrane domain, indicating that VECA may have important biological functions. The present paper describes a procedure for obtaining and purifying human recombinant VECA expressed in Escherichia coli as a fusion protein, via a human VECA cDNA linked pQE30 expression vector to DNA coding for hexahistidine. The purified protein was used to raise anti-(human VECA) polyclonal antibodies, which were suitable for detecting the presence of VECA in cells, cell-culture supernatant and tissues by immunoblotting and immunohistochemistry. To our knowledge, this is the first study on the protein expression and polyclonal-antibody production for human VECA. In addition, we report for the first time the positive identification of VECA in humans at the protein and subcellular level and provide the first experimental verification that VECA was indeed a secreted protein. The anti-(human VECA) polyclonal antibodies prepared may serve as a useful tool for future biological function studies on VECA.
- Published
- 2009
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9. CHROMATOGRAPHIC STUDIES ON AMINOACYL-ACCEPTOR RNA USING PAPER SHEETS OF DIETHYLAMINOETHYL-CELLULOSE
- Author
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Susumu Nishimura and K. Bruce Jacobson
- Subjects
Salt (chemistry) ,Sodium Chloride ,Biochemistry ,Analytical Chemistry ,chemistry.chemical_compound ,Adsorption ,Leucine ,Escherichia coli ,Organic chemistry ,RNA, Messenger ,Cellulose ,chemistry.chemical_classification ,Carbon Isotopes ,Chromatography ,Research ,Organic Chemistry ,RNA ,Proteins ,General Medicine ,Acceptor ,RNA, Bacterial ,chemistry ,Diethylaminoethyl cellulose ,Urea - Abstract
Conditions for the chromatography of amino acyl-acceptor RNA on paper sheets of DEAE-cellulose have been explored further. Amino acid-specific RNA's can be separated using this type of chromatography. It was found that significant amounts of RNA are bound to the paper so that (I) salt solutions were ineffective eluents of RNA, and (2) urea combined with salt was effective. Results suggest that non-electrostatic forces play a significant role in the adsorption of RNA to substituted cellulose.
- Published
- 1964
10. A novel human homologue of the SH3BGR gene encodes a small protein similar to Glutaredoxin 1 of Escherichia coli.
- Author
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Mazzocco M, Arrigo P, Egeo A, Maffei M, Vergano A, Di Lisi R, Ghiotto F, Ciccone E, Cinti R, Ravazzolo R, and Scartezzini P
- Subjects
- Amino Acid Sequence, Animals, Bacterial Proteins chemistry, Bacterial Proteins genetics, Base Sequence, COS Cells, Cell Line, Chlorocebus aethiops, Chromosome Mapping, Cloning, Molecular, Conserved Sequence, Glutaredoxins, Humans, Jurkat Cells, Mice, Molecular Sequence Data, Muscle Proteins chemistry, Organ Specificity, Proteins chemistry, Reverse Transcriptase Polymerase Chain Reaction, Sequence Alignment, Sequence Homology, Amino Acid, T-Lymphocytes, Thioredoxins chemistry, Thioredoxins genetics, Transcription, Genetic, Transfection, Tumor Cells, Cultured, Chromosomes, Human, Pair 1, Escherichia coli genetics, Muscle Proteins genetics, Oxidoreductases, Proteins genetics
- Abstract
Glutaredoxins (GRXs) are ubiquitous GSH-dependent oxidoreductases, which catalyze the reduction of protein-glutathionyl-mixed disulfides and are considered to play an important role in the enzymatic regulation of redox-sensitive proteins. In this paper, we describe the identification and characterization of a new human homologue of the SH3BGR gene, named SH3BGRL3 (SH3 domain binding glutamic acid-rich protein like 3). SH3BGRL3 is widely expressed and codes for a highly conserved small protein, which shows a significant similarity to Glutaredoxin 1 (GRX1) of Escherichia coli and is predicted to belong to the Thioredoxin Superfamily. However, the SH3BGRL3 protein lacks both the conserved cysteine residues, which characterize the enzymatic active site of GRX. This structural feature raises the possibility that SH3BGRL3 could function as an endogenous modulator of GRX biological activity. EGFP-SH3BGRL3 fusion protein expressed in COS-7 cells localizes both to the nucleus and to the cytoplasm. The SH3BGRL3 gene was mapped to chromosome 1p34.3-35., (Copyright 2001 Academic Press.)
- Published
- 2001
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11. Solubility-Weighted Index: fast and accurate prediction of protein solubility
- Author
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Paul P. Gardner, Chun Shen Lim, and Bikash K. Bhandari
- Subjects
Statistics and Probability ,Web server ,Source code ,AcademicSubjects/SCI01060 ,Computer science ,media_common.quotation_subject ,computer.software_genre ,Biochemistry ,Protein expression ,law.invention ,Set (abstract data type) ,03 medical and health sciences ,0302 clinical medicine ,law ,Code (cryptography) ,Escherichia coli ,Solubility ,Molecular Biology ,030304 developmental biology ,media_common ,Mathematics ,0303 health sciences ,Computers ,A protein ,Proteins ,Original Papers ,Computer Science Applications ,Computational Mathematics ,Computational Theory and Mathematics ,Recombinant protein production ,Recombinant DNA ,Stock price index ,Data mining ,Protein solubility ,Biological system ,computer ,Sequence Analysis ,030217 neurology & neurosurgery ,Software - Abstract
MotivationRecombinant protein production is a widely used technique in the biotechnology and biomedical industries, yet only a quarter of target proteins are soluble and can therefore be purified.ResultsWe have discovered that global structural flexibility, which can be modeled by normalised B-factors, accurately predicts the solubility of 12,216 recombinant proteins expressed in Escherichia coli. We have optimised B-factors, and derived a new set of values for solubility scoring that further improves prediction accuracy. We call this new predictor the ‘Solubility-Weighted Index’ (SWI). Importantly, SWI outperforms many existing protein solubility prediction tools. Furthermore, we have developed ‘SoDoPE’ (Soluble Domain for Protein Expression), a web interface that allows users to choose a protein region of interest for predicting and maximising both protein expression and solubility.AvailabilityThe SoDoPE web server and source code are freely available at https://tisigner.com/sodope and https://github.com/Gardner-BinfLab/TISIGNER-ReactJS, respectively. The code and data for reproducing our analysis can be found at https://github.com/Gardner-BinfLab/SoDoPE_paper2020.
- Published
- 2020
12. Toxicity of expanded polyglutamine-domain proteins in Escherichia coli.
- Author
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Onodera O, Roses AD, Tsuji S, Vance JM, Strittmatter WJ, and Burke JR
- Subjects
- Amino Acid Sequence, Cloning, Molecular, Escherichia coli genetics, Glutathione Transferase chemistry, Humans, Molecular Sequence Data, Proteins chemistry, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins toxicity, Escherichia coli metabolism, Peptides chemistry, Proteins toxicity
- Abstract
Five neurodegenerative diseases are caused by proteins with expanded polyglutamine domains. Toxicity of these proteins has been previously identified only in mammals, and no simple model systems are available. In this paper, we demonstrate in E. coli that long polyglutamine domains (59-81 residues) as GST-fusion proteins inhibit growth while smaller glutamine (10-35 residues) or polyalanine (61 residues) domains have no effect. Analogously in humans, polyglutamine repeats less than 35-40 glutamines produce a normal phenotype, while expansion greater than 40 glutamines is always associated with disease. Expression of polyglutamine proteins in E. coli may help identify the molecular mechanism of pathogenesis of CAG trinucleotide repeat diseases and be a useful screen to identify potential therapeutic compound.
- Published
- 1996
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13. GrpE alters the affinity of DnaK for ATP and Mg2+. Implications for the mechanism of nucleotide exchange.
- Author
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Skowyra D and Wickner S
- Subjects
- Bacterial Proteins pharmacology, DNA Replication, Heat-Shock Proteins pharmacology, Kinetics, Adenosine Triphosphatases metabolism, Adenosine Triphosphate metabolism, Bacterial Proteins metabolism, DNA Helicases, DNA-Binding Proteins, Escherichia coli metabolism, Escherichia coli Proteins, HSP70 Heat-Shock Proteins metabolism, Heat-Shock Proteins metabolism, Magnesium metabolism, Proteins, Trans-Activators
- Abstract
DnaK, DnaJ, and GrpE heat shock proteins of Escherichia coli activate site-specific DNA binding by the RepA replication initiator protein of plasmid P1 in a reaction dependent on ATP and Mg2+. We previously showed that GrpE is essential for in vitro RepA activation specifically at about 1 microM free Mg2+. In this paper, we demonstrate that GrpE lowers the requirement of DnaK ATPase for Mg2+, resulting in a large stimulation of ATP hydrolysis at about 1 microM Mg2+ with and without DnaJ and RepA. In contrast to its effect on the Mg2+ requirement, GrpE increases the ATP requirement for DnaK ATPase and dramatically lowers the affinity of DnaK for ATP in the absence of Mg2+. We propose that GrpE not only lowers the affinity of DnaK for nucleotide but, by increasing affinity of DnaK for Mg2+, also weakens the interactions of Mg2+ with nucleotide prior to its release.
- Published
- 1995
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14. Two additional glutaredoxins exist in Escherichia coli: glutaredoxin 3 is a hydrogen donor for ribonucleotide reductase in a thioredoxin/glutaredoxin 1 double mutant.
- Author
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Aslund F, Ehn B, Miranda-Vizuete A, Pueyo C, and Holmgren A
- Subjects
- Amino Acid Sequence, Bacterial Proteins metabolism, Chromatography, Affinity, Chromatography, DEAE-Cellulose, Chromatography, Gel, Deoxyribonucleotides metabolism, Electrophoresis, Polyacrylamide Gel, Escherichia coli genetics, Glutaredoxins, Kinetics, Molecular Sequence Data, Molecular Weight, NAD metabolism, NADP metabolism, Oxidation-Reduction, Proteins isolation & purification, Ribonucleotide Reductases biosynthesis, Ribonucleotide Reductases isolation & purification, Substrate Specificity, Escherichia coli metabolism, Mutation, Oxidoreductases, Proteins metabolism, Ribonucleotide Reductases metabolism
- Abstract
Thioredoxin (Trx) and glutaredoxin (Grx1) are hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. The viability of a double mutant lacking both Trx and Grx1 implies the presence of a third, unknown hydrogen donor. This paper reports the purification and characterization of two proteins with glutaredoxin activity (using hydroxyethyl disulfide as a substrate) from an Escherichia coli mutant lacking Trx and Grx1 (delta trxA, grx::kan). Affinity chromatography was used to bind glutaredoxin on a glutathione-containing thiol-Sepharose column. The molecular weight of Grx2, 27,000, was atypical for glutaredoxins, whereas Grx3 had a molecular weight of 10,000. Amino acid sequence analysis revealed novel structures with putative active sites typical of glutaredoxins: Cys-Pro-Tyr-Cys. The proteins are therefore referred to as Grx2 and Grx3. The low hydrogen donor activity for ribonucleotide reductase in the crude extract was recovered in the purification of Grx3, whereas Grx2 was inactive. As a hydrogen donor for E. coli ribonucleotide reductase, Grx3 showed approximately the same Km value (0.35 microM) as Grx1, whereas its Vmax value was only 5% that of Grx1. The combination of the Grx3 hydrogen donor activity and a 25-fold induction of ribonucleotide reductase activity in a delta trxA, grx double mutant provides an explanation for its viability and deoxyribonucleotide biosynthesis. The physiological functions of Grx2 and Grx3 remain to be determined.
- Published
- 1994
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15. Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth.
- Author
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Janosi L, Shimizu I, and Kaji A
- Subjects
- Bacterial Proteins biosynthesis, Bacterial Proteins genetics, Base Composition, Base Sequence, Chromosomes, Bacterial physiology, Codon, DNA Primers, Escherichia coli genetics, Escherichia coli growth & development, Kinetics, Molecular Sequence Data, Mutagenesis, Plasmids, Ribosomal Proteins, Temperature, Bacterial Proteins metabolism, Escherichia coli metabolism, Genes, Bacterial, Proteins, Ribosomes metabolism
- Abstract
Ribosome releasing factor, product of the frr gene in Escherichia coli, is responsible for dissociation of ribosomes from mRNA after the termination of translation. It functions to "recycle" ribosomes and is renamed ribosome recycling factor in this paper. An E. coli strain was constructed (MC1061-2), which carried frame-shifted frr in the chromosome and wild-type frr on a temperature-sensitive plasmid. MC1061-2 is temperature-sensitive in its growth and does not segregate its frr-carrying plasmid under the plasmid incompatibility pressure. In contrast, isogenic E. coli carrying wild-type frr in the chromosome and mutated frr on the temperature-sensitive plasmid is not temperature-sensitive in its growth and segregates its plasmid from incompatible plasmids. All spontaneously formed thermoresistant colonies derived from MC1061-2 carried wild-type frr that resided either in the bacterial chromosome by re-exchange or in the plasmid, which became temperature-resistant. These observations establish that frr is an essential gene for cell growth.
- Published
- 1994
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16. ExbB acts as a chaperone-like protein to stabilize TonB in the cytoplasm.
- Author
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Karlsson M, Hannavy K, and Higgins CF
- Subjects
- Amino Acid Sequence, Biological Transport, Chaperonins, Energy Metabolism, Molecular Sequence Data, Protein Processing, Post-Translational, Recombinant Fusion Proteins metabolism, Bacterial Proteins metabolism, Cytoplasm metabolism, Escherichia coli metabolism, Escherichia coli Proteins, Membrane Proteins metabolism, Protein Conformation, Proteins metabolism
- Abstract
The TonB protein is required to transduce energy from the cytoplasmic membrane to outer membrane transport proteins of Gram-negative bacteria. Two accessory proteins, ExbB and ExbD, are required for TonB function and it has been suggested that TonB and ExbBD form a complex in the membrane. In this paper we demonstrate that there are two spatially distinct, functional interactions between ExbBD and TonB. First, there is an interaction between ExbBD and the N-terminal signal-like peptide of TonB, probably the formation of a stable complex in the membrane. Second, ExbB interacts with TonB in the cytoplasm. This interaction involves the domain of TonB that is normally periplasmic. Thus, this is a transient interaction which occurs during the synthesis and/or localization of TonB, implying a chaperone-like role for ExbB. The transmembrane topology of ExbB was shown to be consistent with this role.
- Published
- 1993
- Full Text
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17. The Escherichia coli chaperones involved in DNA replication.
- Author
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Zylicz M
- Subjects
- Bacterial Proteins genetics, Chaperonins, Escherichia coli genetics, Genes, Bacterial, HSP40 Heat-Shock Proteins, Heat-Shock Proteins genetics, Bacterial Proteins metabolism, DNA Replication, Escherichia coli metabolism, Escherichia coli Proteins, HSP70 Heat-Shock Proteins, Heat-Shock Proteins metabolism, Proteins metabolism
- Abstract
Mutations in the Escherichia coli heat shock genes, dnaK, dnaJ or grpE, alter host DNA and RNA synthesis, degradation of other proteins, cell division and expression of other heat shock genes. They also block the initiation of DNA replication of bacteriophages lambda and P1, and the mini-F plasmid. An in vitro lambda DNA replication system, composed entirely of purified components, enabled us to describe the molecular mechanism of the dnaK, dnaJ and grpE gene products. DnaK, the bacterial hsp 70 homologue, releases lambda P protein from the preprimosomal complex in an ATP- and DnaJ-dependent reaction (GrpE-independent initiation of lambda DNA replication). In this paper, I show that, when GrpE is present, lambda P protein is not released from the preprimosomal complex, rather it is translocated within the complex in such a way that it does not inhibit DnaB helicase activity. Translocation of lambda P triggers the initiation event allowing DnaB helicase to unwind DNA near the ori lambda sequence, leading to efficient lambda DNA replication. Chaperone activity of the DnaK-DnaJ-GrpE system is first manifested in the selective binding of these heat shock proteins to the preprimosomal complex, followed by its ATP-dependent rearrangement. I show that DnaJ not only tags the preprimosomal complex for recognition by DnaK, but also stabilizes the multi-protein structure. GrpE also participates in the binding of DnaK to the preprimosomal complex by increasing DnaK's affinity to those lambda P proteins which are already with DnaJ.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1993
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18. Differential binding of wild-type and a mutant RepA protein to oriR sequence suggests a model for the initiation of plasmid R1 replication.
- Author
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Giraldo R and Díaz R
- Subjects
- Bacterial Proteins isolation & purification, Base Sequence, Binding Sites, DNA, Bacterial genetics, Deoxyribonuclease I pharmacology, Free Radicals, Guanidine, Guanidines pharmacology, Hydroxides, Macromolecular Substances, Models, Molecular, Molecular Conformation, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, Nucleoproteins drug effects, Nucleoproteins metabolism, Plasmids genetics, Protein Denaturation drug effects, Sequence Homology, Nucleic Acid, Solubility, Bacterial Proteins metabolism, DNA Helicases, DNA Replication, DNA, Bacterial metabolism, DNA-Binding Proteins, Escherichia coli genetics, Plasmids metabolism, Proteins, Trans-Activators
- Abstract
DNA replication of the enterobacterial plasmid R1 is initiated by RepA protein. We have developed a new procedure for the purification of RepA from inclusion bodies, which involves CHAPS-mediated solubilization. This method has been also used for the thermosensitive mutant protein RepA2623. The nucleoprotein complexes obtained with both proteins and oriR, the origin of replication, are studied in this paper. DNaseI and hydroxyl-radical footprinting suggest the presence in oriR of two sites with different affinity for RepA separated by eight helical turns. The pattern of hypersensitive sites in the footprints indicates that the oriR sequence, when complexed with RepA, is curved. The binding of RepA molecules to oriR is co-operative and this co-operativity is defective in the thermosensitive protein. Band-shift analysis of RepA-oriR complexes revealed the existence of a species with an anomalously high electrophoretic mobility that appears after formation of the first RepA-oriR complex and requires the sequential interaction of RepA with its two distal binding sites. These features lead us to propose that protein-protein interactions between RepA bound to both distal sites could be responsible for oriR looping. This model represents a novel mechanism that results in activation of an origin in a replicon that does not contain iterons.
- Published
- 1992
- Full Text
- View/download PDF
19. Structural and functional characterization of the mutant Escherichia coli glutaredoxin (C14----S) and its mixed disulfide with glutathione.
- Author
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Bushweller JH, Aslund F, Wüthrich K, and Holmgren A
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Base Sequence, Disulfides chemistry, Disulfides metabolism, Escherichia coli genetics, Glutaredoxins, Isoelectric Focusing, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligodeoxyribonucleotides, Protein Conformation, Proteins genetics, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Escherichia coli metabolism, Glutathione metabolism, Oxidoreductases, Proteins chemistry, Proteins metabolism
- Abstract
Glutaredoxin is essential for the glutathione (GSH)-dependent synthesis of deoxyribonucleotides by ribonucleotide reductase, and in addition, it displays a general GSH disulfide oxidoreductase activity. In Escherichia coli glutaredoxin, the active site contains a redox-active disulfide/dithiol of the sequence Cys11-Pro12-Tyr13-Cys14. In this paper, we have prepared and characterized the Cys14----Ser mutant of E. coli glutaredoxin and its mixed disulfide with glutathione. The Cys14----Ser mutant of glutaredoxin is shown to retain 38% of the GSH disulfide oxidoreductase activity of the wild-type protein with hydroxyethyl disulfide as substrate but to be completely inactive with ribonucleotide reductase, demonstrating that dithiol glutaredoxin is the hydrogen donor for ribonucleotide reductase. The covalent structure of the mixed disulfide of glutaredoxin(C14S) with GSH prepared with 15N-labeling of the protein was confirmed with nuclear magnetic resonance (NMR) spectroscopy, establishing a basis for NMR structural studies of the glutathione binding site on glutaredoxin.
- Published
- 1992
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20. Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or beta-galactosidase fused to the Hly C-terminal signal domain.
- Author
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Kenny B, Haigh R, and Holland IB
- Subjects
- Amino Acid Sequence, Bacterial Proteins biosynthesis, Base Sequence, Chimera, Chloramphenicol O-Acetyltransferase biosynthesis, Chymosin biosynthesis, Cloning, Molecular, Enzyme Precursors biosynthesis, Membrane Proteins genetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Plasmids, Protein Sorting Signals metabolism, Proteins metabolism, Recombinant Fusion Proteins biosynthesis, Restriction Mapping, beta-Galactosidase biosynthesis, Bacterial Proteins genetics, Chloramphenicol O-Acetyltransferase genetics, Chymosin genetics, Enzyme Precursors genetics, Escherichia coli genetics, Escherichia coli Proteins, Hemolysin Proteins genetics, Protein Processing, Post-Translational, Protein Sorting Signals genetics, Proteins genetics, beta-Galactosidase genetics
- Abstract
Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA, when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the beta-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain.
- Published
- 1991
- Full Text
- View/download PDF
21. Structural and functional comparison between the stability systems ParD of plasmid R1 and Ccd of plasmid F.
- Author
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Ruiz-Echevarría MJ, de Torrontegui G, Giménez-Gallego G, and Díaz-Orejas R
- Subjects
- Amino Acid Sequence, Bacterial Proteins antagonists & inhibitors, Bacterial Toxins antagonists & inhibitors, DNA Replication, DNA, Bacterial biosynthesis, Escherichia coli genetics, Genetic Complementation Test, Genotype, Kinetics, Molecular Sequence Data, Mutation, Sequence Alignment, Bacterial Proteins metabolism, Bacterial Toxins metabolism, DNA Helicases, DNA-Binding Proteins, Escherichia coli physiology, F Factor, Proteins, R Factors, Trans-Activators
- Abstract
The stability determined by the systems ParD of plasmid R1 and Ccd of plasmid F is due to the concerted action of two proteins, a cytotoxin and an antagonist of this function. In this paper we report that CcdA and Kis proteins, the antagonists of the Ccd and ParD systems respectively, share significant sequence homologies at both ends. In Kis, these regions seem to correspond to two different domains. Despite the structural similarities, Kis and CcdA are not interchangeable. In addition we have shown that the cytotoxins of these systems, the Kid and CcdB proteins, do not share structural homologies. In contrast to CcdB, the Kid protein of the ParD system induces RecA-dependent cleavage of the cI repressor of bacteriophage lambda very inefficiently or not at all. The functional implications of these results are discussed.
- Published
- 1991
- Full Text
- View/download PDF
22. In situ synthesis of photoluminescence-quenching nanopaper for rapid and robust detection of pathogens and proteins
- Author
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Songbai Tian, Zhike He, Xinghu Ji, Wang Wang, Yucheng Liu, Guobin Mao, and Meng Liu
- Subjects
Paper ,In situ ,Materials science ,Aptamer ,Nanotechnology ,010402 general chemistry ,01 natural sciences ,Catalysis ,Robustness (computer science) ,Quantum Dots ,Escherichia coli ,Materials Chemistry ,Photoluminescence quenching ,Quenching (fluorescence) ,010405 organic chemistry ,Mucin-1 ,Metals and Alloys ,Cytochromes c ,Proteins ,DNA ,General Chemistry ,Aptamers, Nucleotide ,Fluorescence ,Nanostructures ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Spectrometry, Fluorescence ,Ceramics and Composites - Abstract
A green and facile method is presented for in situ synthesis of a novel photoluminescence-quenching nanopaper with a highly-efficient quenching ability, rapid reaction time and long-term storage. The as-prepared nanopaper is further used to construct an aptasensor platform with high performance, rapidness and robustness.
- Published
- 2019
23. DUckCov: a Dynamic Undocking‐based Virtual Screening Protocol for Covalent Binders
- Author
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András Perczel, György M. Keserű, Xavier Barril, Andrea Scarpino, Moira Rachman, Gyula Pálfy, Dávid Bajusz, and István Vida
- Subjects
Cell Survival ,Protein Conformation ,Computer science ,Covalent binding ,Apoptosis ,01 natural sciences ,Biochemistry ,Drug design ,Cell Line ,Proto-Oncogene Proteins p21(ras) ,Small Molecule Libraries ,Structure-Activity Relationship ,dynamic undocking ,Drug Discovery ,Escherichia coli ,Humans ,Enzyme Inhibitors ,General Pharmacology, Toxicology and Pharmaceutics ,Pharmacology ,Virtual screening ,Disseny de medicaments ,Binding Sites ,Full Paper ,010405 organic chemistry ,Organic Chemistry ,Janus Kinase 3 ,Proteins ,Enzyme inhibitors ,Full Papers ,virtual screening ,covalent docking ,Combinatorial chemistry ,Recombinant Proteins ,Chemical space ,0104 chemical sciences ,Molecular Docking Simulation ,010404 medicinal & biomolecular chemistry ,Inhibidors enzimàtics ,Covalent bond ,Docking (molecular) ,targeted covalent inhibitors ,Molecular Medicine ,Proteïnes ,Software ,Protein Binding - Abstract
Thanks to recent guidelines, the design of safe and effective covalent drugs has gained significant interest. Other than targeting non‐conserved nucleophilic residues, optimizing the noncovalent binding framework is important to improve potency and selectivity of covalent binders toward the desired target. Significant efforts have been made in extending the computational toolkits to include a covalent mechanism of protein targeting, like in the development of covalent docking methods for binding mode prediction. To highlight the value of the noncovalent complex in the covalent binding process, here we describe a new protocol using tethered and constrained docking in combination with Dynamic Undocking (DUck) as a tool to privilege strong protein binders for the identification of novel covalent inhibitors. At the end of the protocol, dedicated covalent docking methods were used to rank and select the virtual hits based on the predicted binding mode. By validating the method on JAK3 and KRas, we demonstrate how this fast iterative protocol can be applied to explore a wide chemical space and identify potent targeted covalent inhibitors.
- Published
- 2019
24. Forthcoming Papers.
- Subjects
- *
PROTEINS , *BIOMOLECULES , *MEMBRANE proteins , *BIOLOGICAL membranes , *ESCHERICHIA coli , *MEDICAL sciences - Abstract
The article presents information on various research papers which will be published in the forthcoming issue of the "European Journal of Biochemistry". Some of the papers discusses are "Enzymatic Synthesis of Neolactotetraosylceramide by the N-Acctyllactosamine Synthase of Human Serum". "Rate of Transplantation and Kinetics of Processing of Newly Synthesized Molecules of Two Major Outer-Membrane Proteins, the OmpA and OmpF Proteins of Escherichia Coli," by the researchers I. Crowlesmith and K. Gamon.
- Published
- 1982
25. Data-collection strategy for challenging native SAD phasing
- Author
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Vincent Olieric, Dianfan Li, Meitian Wang, Tobias Weinert, Natacha Olieric, Aaron D. Finke, Michel O. Steinmetz, Martin Caffrey, Martin Jinek, Carolin Anders, Camelia N. Borca, University of Zurich, and Olieric, Vincent
- Subjects
0301 basic medicine ,Systematic error ,Models, Molecular ,Diacylglycerol Kinase ,Protein Conformation ,Staphylococcus ,Combined use ,CRISPR-Associated Proteins ,610 Medicine & health ,02 engineering and technology ,Biology ,High multiplicity ,Crystallography, X-Ray ,03 medical and health sciences ,1315 Structural Biology ,native SAD phasing ,Structural Biology ,Tubulin ,10019 Department of Biochemistry ,Escherichia coli ,Animals ,membrane protein ,Tubulin complex ,Data collection ,Anomalous diffraction ,Proteins ,DNA ,021001 nanoscience & nanotechnology ,Phaser ,Research Papers ,Rats ,Crystallography ,Multiple data ,data-collection strategy ,030104 developmental biology ,570 Life sciences ,biology ,Stathmin ,Cattle ,0210 nano-technology ,Algorithm ,anomalous signal ,complex ,RNA, Guide, Kinetoplastida - Abstract
The successful structure solution of the integral membrane diacylglycerol kinase and the CRISPR-associated endonuclease RNA–DNA complex by native SAD phasing is demonstrated. The structures were solved with a combined low-dose multi-orientation, multi-crystal data-collection strategy., Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space group C2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.
- Published
- 2016
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- View/download PDF
26. A single amino acid mutation affects elicitor and expansins-like activities of cerato-platanin, a non-catalytic fungal protein
- Author
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Federica Martellini, Francesco Bemporad, Paolo De Paoli, Simone Luti, Luigia Pazzagli, and Lorenzo Mazzoli
- Subjects
0106 biological sciences ,0301 basic medicine ,Fungal Structure ,Mutant ,lcsh:Medicine ,Plant Science ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,chemistry.chemical_compound ,Filter Paper ,Urea ,lcsh:Science ,Plant Proteins ,Fungal protein ,Multidisciplinary ,Organic Compounds ,Cerato-platanin ,Biological activity ,Plants ,Recombinant Proteins ,Elicitor ,Laboratory Equipment ,Chemistry ,Experimental Organism Systems ,Plant Physiology ,Host-Pathogen Interactions ,Physical Sciences ,Engineering and Technology ,Research Article ,Arabidopsis Thaliana ,Equipment ,Brassica ,Mycology ,Biology ,Research and Analysis Methods ,Fungal Proteins ,03 medical and health sciences ,Expansin ,Model Organisms ,Plant and Algal Models ,Phytoalexins ,medicine ,Escherichia coli ,Plant Defenses ,Cellulose ,lcsh:R ,Pathogen-Associated Molecular Pattern Molecules ,Organic Chemistry ,Chemical Compounds ,Organisms ,Fungi ,Biology and Life Sciences ,Proteins ,Plant Disease Resistance ,Plant Pathology ,030104 developmental biology ,chemistry ,Amino Acid Substitution ,cerato-platanin ,defence elicitor, E. coli SHuffle, expansin-like protein, PAMP, ROS ,lcsh:Q ,010606 plant biology & botany - Abstract
Cerato-platanin (CP) is a non-catalytic, cysteine-rich protein, the first member of the cerato-platanin family. It is a single-domain protein with a double Ψ/β barrel domain resembling the D1 domain of plant and bacterial expansins. Similarly to expansins, CP shows a cell wall-loosening activity on cellulose and can be defined as an expanisin-like protein, in spite of the missing D2 domain, normally present in plant expansins. The weakening activity shown on cellulose may facilitate the CP-host interaction, corroborating the role of CP in eliciting plant defence response. Indeed, CP is an elicitor of primary defences acting as a Pathogen-Associated Molecular Patterns (PAMP). So far, structure-function relationship study has been mainly performed on the bacterial BsEXLX1 expansin, probably due to difficulties in expressing plant expansins in heterologous systems. Here, we report a subcloning and purification method of CP in the engineered E. coli SHuffle cells, which proved to be suitable to obtain the properly folded and biologically active protein. The method also enabled the production of the mutant D77A, rationally designed to be inactive. The wild-type and the mutated CP were characterized for cellulose weakening activity and for PAMP activity (i.e. induction of Reactive Oxygen Species synthesis and phytoalexins production). Our analysis reveals that the carboxyl group of D77 is crucial for expansin-like and PAMP activities, thus permitting to establish a correlation between the ability to weaken cellulose and the capacity to induce defence responses in plants. Our results enable the structural and functional characterization of a mono-domain eukaryotic expansin and identify the essential role of a specific aspartic residue in cellulose weakening.
- Published
- 2016
27. The role of the codon and the initiation factor IF-2 in the selection of N-blocked aminoacyl-tRNA for initiation.
- Author
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van der Laken K, Bakker-Steeneveld H, Berkhout B, and van Knippenberg PH
- Subjects
- Eukaryotic Initiation Factor-2, Kinetics, Polyribonucleotides metabolism, Ribosomes metabolism, Structure-Activity Relationship, Codon, Escherichia coli metabolism, Peptide Initiation Factors metabolism, Proteins metabolism, RNA, Messenger, RNA, Transfer, Amino Acyl metabolism
- Abstract
Poly(uridylic acid) [poly(U)] and poly(xanthidylic acid) [poly(X)] strongly stimulate the IF-2-dependent binding of fMET-tRNA to 30-S ribosomal subunits from Escherichia coli [Van der Laken et al. (1979) FEBS Lett. 100, 230-234]. The N-formylmethionine moiety is incorporated into poly(phenylalanine) upon subsequent addition of other components required for protein synthesis when poly(U) is used as template. This paper shows that N-acetylated Phe-tRNAPhe (AcPhe-tRNA), but not Phe-tRNAPhe or tRNAPhe, competes with fMET-tRNA for binding to poly(U)-programmed 30-S ribosomal subunits. The two species of N-blocked aminoacyl-tRNA are bound to poly(U)-programmed and poly(X)-programmed 30-S subunits in a ratio that is linearly dependent on the ratio of the two species added. With poly(U) as template there is no apparent preference for either fMET-tRNA or AcPhe-tRNA, whereas with poly(X) there is a 2-3-fold preference for fMET-tRNA. The initiation factor IF-2, which is strictly required for the binding of N-blocked aminoacyl-tRNAs, has a higher affinity for fMET-tRNA than for AcPhe-tRNA. It is concluded that (a) interaction of the 30-S ribosomal subunit with poly(U) or poly(X) leads to IF-2-dependent binding of N-blocked aminoacyl-tRNA; (b) the initiation factor IF-2-discriminates in favour of fMET-TRNA; (c) the presence of the cognate codon discriminates in favour of the corresponding N-blocked aminoacyl-tRNA.
- Published
- 1980
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28. Phosphoproteins in Escherichia coli.
- Author
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RAFTER GW and LANE WC
- Subjects
- Bacterial Proteins, Escherichia coli metabolism, Phosphoproteins, Proteins metabolism
- Abstract
Rafter, Gale W. (The Johns Hopkins School of Medicine and School of Hygiene and Public Health, Baltimore, Md.) and William C. Lane. Phosphoproteins in Escherichia coli. J. Bacteriol. 83:1077-1083. 1962.-The identification and metabolism of phosphoprotein were investigated in Escherichia coli. Hydrolysis of bacterial protein fractions with barium hydroxide or with phosphoprotein phosphatase released acid-soluble phosphorus. Chromatography of acid-hydrolyzed and incubated fractions also indicated the presence of phospho-amino acids. Turnover of phosphate in protein of growing cells was not observed, but incorporation of phosphate into protein of nongrowing cells was found. The protein-phosphate content decreased as organisms passed from the growing to the nongrowing state. The phosphoprotein composition, as revealed by paper electrophoresis, was heterogeneous. No protein phosphokinase or protein-phosphate phosphatase was detected in cell-free extracts, but an active principle which catalyzed the formation of acid-soluble phosphate from bacterial protein fractions was found.
- Published
- 1962
- Full Text
- View/download PDF
29. The natural occurrence of ethionine in bactdria.
- Author
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FISHER JF and MALLETTE MF
- Subjects
- Animals, Cattle, Bacteria metabolism, Culture Media, Escherichia coli metabolism, Ethionine metabolism, Methionine, Proteins
- Abstract
Two unknown radioactive areas appeared after radioautography and two dimensional paper chromatography of culture medium in which Escherichia coli was grown. These materials were studied by paper chromatography and paper electrophoresis of several derivatives and identified as ethionine and ethionine sulfone, the latter an artifact. Chromatographic coincidence of the unknowns and their derivatives with authentic materials establishes the identification. Ethionine was found in cellular extracts and in the growth media of Escherichia coli, Bacillus megaterium, Pseudomonas aeruginosa, and Aerobacter aerogenes but not in Scenedesmus, Saccharomyces cerevisiae, or bovine lymphosarcoma cells. Ethionine was synthesized by resting E. coli cultures from radioactive sulfate and from radioactive methionine. Growing cells labeled ethionine within 1 minute after addition of radioactive sulfate to cultures. Levels of radioactivity in ethionine increased with time. No incorporation of this amino acid could be detected in the cellular proteins formed under the conditions of this study.
- Published
- 1961
- Full Text
- View/download PDF
30. Baking a mass-spectrometry data PIE with McMC and simulated annealing: predicting protein post-translational modifications from integrated top-down and bottom-up data
- Author
-
Morgan C. Giddings and Stuart R. Jefferys
- Subjects
Proteomics ,Ribosomal Proteins ,Statistics and Probability ,Gene isoform ,Computer science ,computer.software_genre ,Mass spectrometry ,Biochemistry ,Mass Spectrometry ,symbols.namesake ,Bacterial Proteins ,Ribosomal protein ,Escherichia coli ,Protein Isoforms ,Molecular Biology ,Markov chain ,Proteins ,A protein ,Experimental data ,Markov chain Monte Carlo ,Original Papers ,Markov Chains ,Computer Science Applications ,Computational Mathematics ,Computational Theory and Mathematics ,Simulated annealing ,Posttranslational modification ,symbols ,Protein inference ,Data mining ,Monte Carlo Method ,Protein Processing, Post-Translational ,computer ,Algorithms ,Software - Abstract
Motivation: Post-translational modifications are vital to the function of proteins, but are hard to study, especially since several modified isoforms of a protein may be present simultaneously. Mass spectrometers are a great tool for investigating modified proteins, but the data they provide is often incomplete, ambiguous and difficult to interpret. Combining data from multiple experimental techniques—especially bottom-up and top-down mass spectrometry—provides complementary information. When integrated with background knowledge this allows a human expert to interpret what modifications are present and where on a protein they are located. However, the process is arduous and for high-throughput applications needs to be automated. Results: This article explores a data integration methodology based on Markov chain Monte Carlo and simulated annealing. Our software, the Protein Inference Engine (the PIE) applies these algorithms using a modular approach, allowing multiple types of data to be considered simultaneously and for new data types to be added as needed. Even for complicated data representing multiple modifications and several isoforms, the PIE generates accurate modification predictions, including location. When applied to experimental data collected on the L7/L12 ribosomal protein the PIE was able to make predictions consistent with manual interpretation for several different L7/L12 isoforms using a combination of bottom-up data with experimentally identified intact masses. Availability: Software, demo projects and source can be downloaded from http://pie.giddingslab.org/ Contact: morgan@giddingslab.org. Supplementary information: Supplementary data are available at Bioinformatics online
- Published
- 2011
31. Expression of a selenomethionyl derivative and preliminary crystallographic studies of human cystatin C
- Author
-
Elżbieta Jankowska, Mariusz Jaskolski, Magnus Abrahamson, Anders Grubb, Marcia Alvarez Fernandez, Zbigniew Grzonka, Robert Janowski, and Maciej Kozak
- Subjects
Proteases ,Stereochemistry ,Molecular Sequence Data ,protease inhibitors ,Cysteine Proteinase Inhibitors ,Crystallography, X-Ray ,medicine.disease_cause ,cysteine proteases ,law.invention ,chemistry.chemical_compound ,Tetragonal crystal system ,Structural Biology ,law ,selenomethionyl derivatives ,Escherichia coli ,medicine ,Humans ,Amino Acid Sequence ,Cystatin C ,Selenomethionine ,Selenoproteins ,biology ,Proteins ,General Medicine ,Crystallization Papers ,Cystatins ,Recombinant Proteins ,Crystallography ,Structural biology ,chemistry ,biology.protein ,Recombinant DNA ,Cystatin ,Crystallization ,Derivative (chemistry) - Abstract
Human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of papain-like mammalian proteases, has been produced in its full-length form by recombinant techniques and crystallized in two polymorphic forms: cubic and tetragonal. A selenomethionyl derivative of the protein, obtained by Escherichia coli expression and with complete Met→Se-Met substitution confirmed by mass spectrometry, amino-acid analysis and X-ray absorption spectra, was crystallized in the cubic form. A truncated variant of the protein, lacking ten N-terminal residues, has also been crystallized. The crystals of this variant are tetragonal and, like the two polymorphs of the full-length protein, contain multiple copies of the molecule in the asymmetric unit, suggesting oligomerization of the protein.
- Published
- 1999
32. Efficient Robust Yield Method for Preparing Bacterial Ghosts by Escherichia coli Phage ID52 Lysis Protein E.
- Author
-
Ma, Yi, Zhu, Wenjun, Zhu, Guanshu, Xu, Yue, Li, Shuyu, Chen, Rui, Chen, Lidan, and Wang, Jufang
- Subjects
ESCHERICHIA coli ,LYSIS ,SALMONELLA typhimurium ,MASS production ,BACTERIOPHAGES ,PROTEINS - Abstract
Bacterial ghosts (BGs) are nonliving empty bacterial shells without cytoplasm retaining original morphology and identical antigenicity of natural bacteria, making them high potential and promising vaccine candidates and delivery vehicles. However, the low yield of commonly used BGs preparation methods limits its mass production and widely application. In order to improve BGs production, E. coli phage ID52 lysis protein E was introduced to generating BGs for the first time. Above all, we compared the lysis activity of lysis protein of E. coli phage φX174 and E. coli phage ID52 as well as the effects of promoters on the lysis activity of ID52-E, which shown that the lysis activity and BGs formation rate of protein ID52-E was significantly higher than protein φX174-E. Further, the lysis activity of ID52-E was significantly improved under the control of L-arabinose inducible promoter which initial induction OD
600 reached as high as 2.0. The applicability of lysis protein ID52-E induced by L-arabinose was proved by preparing probiotic E. coli Nissle 1917 BGs and pathogenic Salmonella typhimurium BGs in mass production. This paper introduced a novel and highly efficient method for BGs preparation depending on recombinant expression of E. coli phage ID52-E under eco-friendly and reasonable price inducer L-arabinose. [ABSTRACT FROM AUTHOR]- Published
- 2022
- Full Text
- View/download PDF
33. It's all in the crystals…
- Author
-
Zygmunt S. Derewenda
- Subjects
Models, Molecular ,Materials science ,crystallization ,Surface Properties ,crystal contacts ,Entropy ,Macromolecular crystallography ,Proteins ,Nanotechnology ,protein engineering ,General Medicine ,Protein engineering ,Crystallography, X-Ray ,Research Papers ,law.invention ,macromolecular crystallography ,Structural Biology ,law ,Mutation ,Escherichia coli ,crystals ,Molecular surfaces ,Crystallization ,Protein crystallization ,Protein Structure, Quaternary - Abstract
Protein surface engineering is increasingly used as a routine tool to enhance the crystallization propensity of proteins. Future possibilities include the use of multi-site protein variants, rational modulation of solubility and the development of strategies to tackle membrane proteins., Macromolecular crystallography relies on the availability and quality of single crystals; these are typically obtained through extensive screening, which has a very low intrinsic success rate. Crystallization is not a completely stochastic process and many proteins do not succumb to crystallization because of specific microscopic features of their molecular surfaces. It follows that rational surface engineering through site-directed mutagenesis should allow a systematic and significant improvement in crystallization success rates. Here, one such established strategy, surface-entropy reduction (SER), is discussed, including its successes, limitations and possible future developments.
- Published
- 2010
34. Design principles of the proteolytic cascade governing the sigmaE-mediated envelope stress response in Escherichia coli: keys to graded, buffered, and rapid signal transduction
- Author
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Rachna Chaba, Tania A. Baker, Irina L. Grigorova, Julia M. Flynn, and Carol A. Gross
- Subjects
Proteases ,Chromatin Immunoprecipitation ,Cytoplasm ,Proteolysis ,Blotting, Western ,Sigma Factor ,Biology ,Cleavage (embryo) ,chemistry.chemical_compound ,Adenosine Triphosphate ,Sigma factor ,Endopeptidases ,Genetics ,medicine ,Escherichia coli ,Animals ,Humans ,ATP-Dependent Proteases ,Transcription factor ,medicine.diagnostic_test ,Escherichia coli Proteins ,Proteins ,Membrane Proteins ,beta-Galactosidase ,Adaptation, Physiological ,Cell biology ,Kinetics ,Biochemistry ,chemistry ,Signal transduction ,Protein Processing, Post-Translational ,Adenosine triphosphate ,Research Paper ,Developmental Biology ,Signal Transduction ,Transcription Factors - Abstract
Proteolytic cascades often transduce signals between cellular compartments, but the features of these cascades that permit efficient conversion of a biological signal into a transcriptional output are not well elucidated. σE mediates an envelope stress response in Escherichia coli, and its activity is controlled by regulated degradation of RseA, a membrane-spanning anti-σ factor. Examination of the individual steps in this protease cascade reveals that the initial, signal-sensing cleavage step is rate-limiting; that multiple ATP-dependent proteases degrade the cytoplasmic fragment of RseA and that dissociation of σE from RseA is so slow that most free σE must be generated by the active degradation of RseA. As a consequence, the degradation rate of RseA is set by the amount of inducing signal, and insulated from the “load” on and activity of the cytoplasmic proteases. Additionally, changes in RseA degradation rate are rapidly reflected in altered σE activity. These design features are attractive as general components of signal transduction pathways governed by unstable negative regulators.
- Published
- 2007
35. Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study
- Author
-
Alois Bonifacio, Cees Gooijer, Gert van der Zwan, Chris de Graaf, Koop Lammertsma, Peter H. J. Keizers, André R. Groenhof, Jan N. M. Commandeur, Nico P. E. Vermeulen, Andreas W. Ehlers, BioAnalytical Chemistry, Organic Chemistry, Molecular and Computational Toxicology, Medicinal chemistry, Bonifacio, Aloi, Groenhof, A., Keizers, P. H. J., De Graaf, C., Commandeur, J. N. M., Vermeulen, N. P. E., Ehlers, A. W., Lammertsma, K., Gooijer, C., and Van Der Zwan, G.
- Subjects
Models, Molecular ,Resonance Raman ,spectroscopy ,Spin states ,Stereochemistry ,enzymes ,Mutant ,Heme ,Crystallography, X-Ray ,Spectrum Analysis, Raman ,Biochemistry ,digestive system ,Inorganic Chemistry ,chemistry.chemical_compound ,Escherichia coli ,Spin equilibrium ,Humans ,Binding site ,skin and connective tissue diseases ,Raman ,T309V ,Original Paper ,Binding Sites ,biology ,Wild type ,Active site ,Computational Biology ,Threonine mutant ,Cytochrome P450 2D6 ,proteins ,enzyme ,chemistry ,Cytochrome P-450 CYP2D6 ,Mutation ,biology.protein ,Density functional theory ,Spectrophotometry, Ultraviolet ,protein ,Plasmids - Abstract
Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant. Electronic supplementary material The online version of this article (doi:10.1007/s00775-007-0210-5) contains supplementary material, which is available to authorized users.
- Published
- 2007
36. ELECTROPHORETIC METHOD FOR OBTAINING NUCLEOTIDES LABELLED WITH RADIOACTIVE CARBON
- Author
-
Berbec, H
- Published
- 1963
- Full Text
- View/download PDF
37. ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification.
- Author
-
Cardoso, Valdemir M., Paredes, Sheyla A. H., Campani, Gilson, Gonçalves, Viviane M., and Zangirolami, Teresa C.
- Subjects
ENDOTOXINS ,RECOMBINANT proteins ,PNEUMOCOCCAL vaccines ,MORPHOLOGY ,PROTEINS ,ESCHERICHIA coli - Abstract
Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-β-d-thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production. Key points: • ClearColi can produce as much PspA4Pro as conventional E. coli BL21(DE3) cells. • 10.5 g PspA4Pro produced in ClearColi bioreactor culture using a defined medium. • Functional PspA4Pro (98% of purity) was obtained in ClearColi bioreactor culture. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
38. The Natural Occurrence of Ethionine in Bacteria
- Author
-
J. F. Fisher and M. F. Mallette
- Subjects
Physiology ,Enterobacter aerogenes ,medicine.disease_cause ,Article ,chemistry.chemical_compound ,Methionine ,Escherichia coli ,medicine ,Animals ,Ethionine ,Bacillus megaterium ,chemistry.chemical_classification ,Chromatography ,Bacteria ,biology ,Proteins ,biology.organism_classification ,Culture Media ,Amino acid ,Paper chromatography ,chemistry ,Biochemistry ,Cattle - Abstract
Two unknown radioactive areas appeared after radioautography and two dimensional paper chromatography of culture medium in which Escherichia coli was grown. These materials were studied by paper chromatography and paper electrophoresis of several derivatives and identified as ethionine and ethionine sulfone, the latter an artifact. Chromatographic coincidence of the unknowns and their derivatives with authentic materials establishes the identification. Ethionine was found in cellular extracts and in the growth media of Escherichia coli, Bacillus megaterium, Pseudomonas aeruginosa, and Aerobacter aerogenes but not in Scenedesmus, Saccharomyces cerevisiae, or bovine lymphosarcoma cells. Ethionine was synthesized by resting E. coli cultures from radioactive sulfate and from radioactive methionine. Growing cells labeled ethionine within 1 minute after addition of radioactive sulfate to cultures. Levels of radioactivity in ethionine increased with time. No incorporation of this amino acid could be detected in the cellular proteins formed under the conditions of this study.
- Published
- 1961
39. DELAY-INDEPENDENT STABILITY OF GENETIC REGULATORY NETWORKS WITH TIME DELAYS.
- Author
-
FANG-XIANG WU
- Subjects
GENES ,PROTEINS ,DIFFERENTIAL equations ,ESCHERICHIA coli ,CELLS - Abstract
In an organism, genes encode proteins, some of which in turn regulate other genes. Such interactions work in highly structured but incredibly complex ways, and make up a genetic regulatory network. Recently, nonlinear delay differential equations have been proposed for describing genetic regulatory networks in the state-space form. In this paper, we study stability properties of genetic regulatory networks with time delays, by the notion of delay-independent stability. We first present necessary and sufficient conditions for delay-independent local stability of genetic regulatory networks with a single time delay, and then extend the main result to genetic regulatory networks with multiple time delays. To illustrate the main theory, we analyze delay-independent stability of three genetic regulatory networks in E. coli or zebra fish. For E. coli, an autoregulatory network and a repressilatory network are analyzed. The results show that these two genetic regulatory networks with parameters in the physiological range are delay-independently robustly stable. For zebra fish, an autoregulatory network for the gene her1 is analyzed. The result shows that delay-independent stability of this network depends on the initial number of protein molecules, which is in agreement with the existing biological knowledge. The theories presented in this paper provide a very useful complement to the previous work and a framework for further studying the stability of more complex genetic regulatory networks. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
40. A Novel Handheld Fluorimeter for Rapid Detection of Escherichia coli in Drinking Water.
- Author
-
Ferrero Martin, Francisco Javier, Valledor Llopis, Marta, Campo Rodriguez, Juan Carlos, Marin Fernandez, Laura, Gutierrez-del-Rio Menendez, Ignacio, Fernandez Fernandez, Javier, Lombo Brugos, Felipe, Cobian Fernandez, Natalia, Olmos Fernandez Corugedo, Francisco, and Mendez Suarez, Irene
- Abstract
The microbiological quality of drinking water is a concern to consumers, water suppliers, regulators, and public health authorities alike. Monitoring the microbiological quality of drinking water largely relies on the examination of indicator bacteria such as coliforms like Escherichia coli. E. coli is widely used as an indicator of fecal pollution when monitoring the microbial quality of drinking water, because it is abundant in all mammal feces and, therefore, is found in sewage and in natural waters contaminated with fecal matter, from human origin, wild animals, or derived from agricultural activity. This paper describes the development of a novel handheld fluorimeter for the rapid detection of E. coli in drinking water based on a specific cellular biomarker. The measurement system is based on a photomultiplier tube that captures the fluorescence signal produced by the cellular biomarker when it is excited by an ultraviolet LED. The cellular biomarker is also developed and it consists of a chimeric protein with a Green fluorescent protein (GFP) in the N-terminal domain and a specific amino acid sequence in the C-terminal domain (Colicin S4) that targets specifically the structure of the microorganism to be detected. The instrument is simple to use, lightweight, and can be powered by either an ac/dc power adapter or a rechargeable battery, making it an excellent choice for the rapid detection of E. coli in drinking water in field studies and laboratory measurements. [ABSTRACT FROM PUBLISHER]
- Published
- 2016
- Full Text
- View/download PDF
41. Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons.
- Author
-
Hu, Kuang, Chou, Chia-Wei, Wilke, Claus O., and Finkelstein, Ilya J.
- Subjects
ESCHERICHIA coli ,GENOME editing ,CRISPRS ,RNA ,PROTEINS - Abstract
CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications. Here, the authors show that CRISPR-associated transposons can co-opt other CRISPR arrays found in the same cell. Such interactions must be considered when porting these systems into new hosts. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
42. Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation.
- Author
-
Gupta, Meera, Johnson, Alex N. T., Cruz, Edward R., Costa, Eli J., Guest, Randi L., Li, Sophia Hsin-Jung, Hart, Elizabeth M., Nguyen, Thao, Stadlmeier, Michael, Bratton, Benjamin P., Silhavy, Thomas J., Wingreen, Ned S., Gitai, Zemer, and Wühr, Martin
- Subjects
ESCHERICHIA coli ,PROTEOLYSIS ,RADIOLABELING ,CELL division ,PROTEINS ,PROTEOLYTIC enzymes - Abstract
Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis. Gupta, Johnson et al. quantify the turnover rates of ~3200 E. coli proteins, demonstrating that cytoplasmic proteins are recycled when nitrogen is limited and that protein degradation rates are generally uncoupled from cell division rates. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
43. Role of the conserved pyridoxal 5'-phosphate-binding protein YggS/PLPBP in vitamin B6 and amino acid homeostasis.
- Author
-
Tomokazu Ito
- Subjects
- *
AMINO acids , *ESCHERICHIA coli , *HOMEOSTASIS , *VITAMINS , *PROTEINS - Abstract
The YggS/PLPBP protein (also called COG0325 or PLPHP) is a conserved pyridoxal 5 '-phosphate (PLP) -binding protein present in all 3 domains of life. Recent studies have demonstrated that disruption or mutation of this protein has multifaceted effects in various organisms, including vitamin B6 -dependent epilepsy in humans. In Escherichia coli, disruption of this protein--encoded by yggS--perturbs Thr-Ile/Val metabolism, one-carbon metabolism, coenzyme A synthesis, and vitamin B6 homeostasis. This protein is critical for maintaining low levels of pyridoxine 5 '-phosphate (PNP) in various organisms. In the yggS -deficient E. coli strain, inhibition of PLP-dependent enzymes, such as the glycine cleavage system by PNP, is the root cause of metabolic perturbation. Our data suggest that the YggS/PLPBP protein may be involved in the balancing of B6 vitamers by mediating efficient turnover of protein-bound B6 vitamers. This paper reviews recent findings on the function of the YggS/PLPBP protein. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
44. 50-S Ribosomal Proteins.
- Subjects
PROTEINS ,ESCHERICHIA coli ,BIOCHEMISTRY ,PEPTIDES ,AMINO acids ,RIBOSOMES - Abstract
Peptide studies on two acidic proteins, A
1 and A2 , from 50-S ribosomes of Escherichia coli indicate a closely related primary structure. 1. Amino acid compositions of the tryptic peptides of the two proteins are identical, with exception of the amino terminal peptide. 2. The N-terminal amino acid sequence of A2 -protein is Ser-Ile-Thr-Lys, while that of A1 -protein is N-acetyl-Ser-Ile-Thr-Lys. 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent with the physical molecular weight estimate. 4. In 50% of the polypeptide chains, in both A1 - or A2 -protein, a specific lysine residue is replaced by ε-N-monomethyl-lysine. [ABSTRACT FROM AUTHOR]- Published
- 1972
45. Cell Envelope Composition of Escherichia coli K12: A Comparison of the Cell Poles and the Lateral Wall
- Author
-
Uli Schwarz, Ernest W. Goodell, and Ron M. Teather
- Subjects
Chromatography, Paper ,Peptidoglycan ,Biology ,Cell Fractionation ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,Cell Wall ,Deoxy Sugars ,Ketoses ,Centrifugation, Density Gradient ,Escherichia coli ,medicine ,Inner membrane ,NADH, NADPH Oxidoreductases ,Phospholipids ,Phosphatidylglycerol ,Strain (chemistry) ,Proteins ,Sugar Acids ,Culture Media ,Succinate Dehydrogenase ,Membrane ,chemistry ,Cytoplasm ,Glycerophosphates ,Isotope Labeling ,Biophysics ,Electrophoresis, Polyacrylamide Gel ,Chromatography, Thin Layer ,Cell envelope ,Bacterial outer membrane ,Subcellular Fractions - Abstract
Minicells and filaments of Escherichia coli strain P678–54 provide a convenient means of preparing cell-envelope fractions that are, respectively, greatly enriched or reduced in material derived from the cell poles. The inner (cytoplasmic) membrane, outer membrane, and sacculus were prepared both from minicells and from filaments purified from a single culture. One protein component and phosphatidylglycerol were found to be enriched in minicell inner membrane.
- Published
- 1974
46. Polypeptide Chain Initiation and Stepwise Elongation with Artemia Ribosomes and Factors
- Author
-
Michael Zasloff, Severo Ochoa, and Ralph P. McCroskey
- Subjects
Peptide Biosynthesis ,Embryo, Nonmammalian ,Formates ,GTP' ,Uracil Nucleotides ,Phenylalanine ,Protein subunit ,Polynucleotides ,Peptide Chain Elongation, Translational ,Acetates ,Biology ,Tritium ,Ribosome ,chemistry.chemical_compound ,Methionine ,RNA, Transfer ,Decapoda ,Escherichia coli ,Animals ,Initiation factor ,Electrophoresis, Paper ,Eukaryotic Small Ribosomal Subunit ,Peptide Chain Initiation, Translational ,Carbon Isotopes ,Multidisciplinary ,Eukaryotic Large Ribosomal Subunit ,Proteins ,Ribonucleotides ,Elongation factor ,Biochemistry ,chemistry ,Puromycin ,Guanosine Triphosphate ,Biological Sciences: Biochemistry ,Ribosomes - Abstract
The supernatant initiation factor from Artemia salina embryos promotes, besides the AUG-dependent binding of fMet-tRNA f , the poly(U)-dependent binding of N -acetylPhe-tRNA to 40S ribosomal subunits; the bound N -acylaminoacyl-tRNA reacts directly with puromycin upon addition of 60S subunits. Both the binding reaction and the synthesis of N -acylaminoacyl-puromycin occur in the absence of GTP or other ribonucleoside triphosphates. To a smaller extent, the factor also mediates the 40S ribosomal binding of Met-tRNA f and Phe-tRNA; in this case, the bound aminoacyl-tRNA is less reactive with puromycin. After the poly(U)- and supernatant factor-dependent binding of N -acetylPhe-tRNA to 40S subunits at low Mg 2+ concentration, binding of a second aminoacyl-tRNA (Phe-tRNA), with ensuing formation of the first peptide bond, is dependent upon the addition of the 60S subunit, elongation factor EF-1, and GTP. Further growth of the polypeptide chain requires translocation and is, therefore, dependent upon the addition of elongation factor EF-2. As with the Escherichia coli system, once requirements for translation of the third codon have been met, no further additions are necessary for elongation of a peptide chain.
- Published
- 1972
47. Nature of the ribosomal binding site for initiation factor 3 (IF-3)
- Author
-
Cynthia L. Pon and Claudio O. Gualerzi
- Subjects
Alkylation ,Phenylalanine ,Biophysics ,Biology ,Biochemistry ,Antigen-Antibody Reactions ,Ribonucleases ,RNA, Transfer ,Peptide Initiation Factors ,23S ribosomal RNA ,Centrifugation, Density Gradient ,Escherichia coli ,Electrophoresis, Paper ,Trypsin ,RNA, Messenger ,Binding site ,Molecular Biology ,50S ,Carbon Isotopes ,Binding Sites ,Immune Sera ,Proteins ,Cell Biology ,Cations, Monovalent ,Ribosomal RNA ,Ribosomal binding site ,DNA binding site ,Kinetics ,A-site ,Protein Biosynthesis ,Acridines ,Ribosomes ,Protein Binding ,Binding domain - Abstract
In vitro labelled IF-3 binds to both 16S and 23S rRNA but while one molecule of IF-3 binds to each 30S particle, binding to 50S particles is negligible. If proteins are removed by LiCl or CsCl treatment from either ribosomal subunit, however, binding specificity is lost and new “binding sites” appear on both ribosomal particles. Controlled RNase digestion of the 30S subunits does not cause the loss of any r-protein while controlled trypsin digestion results in the loss or degradation of several r-proteins; compared to the Phe-tRNA binding site, the binding site of IF-3 seems to be more sensitive to RNase than to trypsin digestion. Antibodies against single 30S r-proteins, which inhibit other ribosomal functions, do not prevent the binding of IF-3. RNA-binding dyes (acridine orange and pyronine) inhibit the binding of IF-3 to 30S ribosomal subunits. It is proposed that a segment of the 16S rRNA provides the binding site for IF-3 and that r-proteins confer specificity, restricting the number of available “binding sites”, and stabilize the 30S-IF-3 interaction.
- Published
- 1973
48. Elastic Image Registration with Applications to Proteomics.
- Author
-
Sorzano, C. O. S., Thévenaz, P., Valdés, I., Beloso, A., and Unser, M.
- Subjects
PROTEOMICS ,POLYACRYLAMIDE gel electrophoresis ,ESCHERICHIA coli ,STOCHASTIC processes ,PROTEINS - Abstract
In proteomics, electrophoretic 2-D gels are used to separate the proteome according to the molecular weight and electrical charge of its constituents, which are proteins expressed by a cell or organ at a particular time and under specific conditions. One of the main applications is the analysis of differential expressions between different conditions for which certain (perhaps many) spots are present in one of the images, but not in the other. One of the difficulties of this analysis is that 2-D gels are affected by spatial distortions due to run-time differences and dye-front deformations, which results in images that are significantly dissimilar and that pose a challenging problem to image-registration algorithms. In this paper, we test the efficiency of a state-of-the-art elastic-registration algorithm that we had already introduced in the context of biomedical images. We study here the registration of simulated 2-D gels with known expression patterns and deformations. We show that our algorithm is capable of handling such situations. The proposed algorithm is publicly available at http://bigwww.epfl.ch under the name UnWarpJ. © 2006 American Institute of Physics [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
49. More pieces in the promoter jigsaw: recognition of −10 regions by alternative sigma factors.
- Author
-
Busby, Stephen J. W.
- Subjects
ESCHERICHIA coli ,BIOMOLECULES ,PROTEINS ,MOLECULAR microbiology ,MICROBIOLOGY ,ENTEROBACTERIACEAE ,BIOCHEMISTRY - Abstract
Two papers in this issue of Molecular Microbiology from Carol Gross and colleagues describe experiments that investigate how two alternative Escherichia coliσ proteins recognize target promoter −10 regions. A combination of genetics, biochemistry and bioinformatics is used to show that determinants in two adjacent domains of the σ proteins contact discrete sequence elements in the −10 regions. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
50. Kinetic Models Demonstrate Ability of Staphylococcus aureus to Uptake Heme from Beta vulgaris Proteins.
- Author
-
Abhishek, Suman, Gupta, Anil Kumar, and Singh, Anjuvan
- Subjects
MICROORGANISMS ,BIOCHEMISTRY ,PATHOGENIC microorganisms ,PROTEINS ,ESCHERICHIA coli - Abstract
Heme, a Fe-centred hexacoordinated organometallic compound mainly present in blood proteins, acts as a major source of iron (Fe2+) for various hemolytic microorganisms. This makes blood an essential mediumsupplement for characterizing such organisms. Considering the increasing blood demand for this purpose, we are proposing an alternative approach for hemolytic characterization of bacteria using plant derivative. For the first time, we present the kinetic model of growth of Staphylococcus aureus, a beta-hemolytic, gram-positive rod-shaped bacterium, and subsequent heme uptake at an optimized concentration of crude proteins of Beta vulgaris (red beetroot). In this paper, we have determined the heme content of the beetroot juice and demonstrated the ability of S. aureus to uptake heme from beetroot protein solution. According to the determined growth kinetics, the doubling time of the bacterium in the beetroot protein supplemented medium was 25 minutes. The heme uptake analysis showed a zeroth order kinetics with a constant decline rate of 0.19 μM h
-1 . [ABSTRACT FROM AUTHOR]- Published
- 2017
- Full Text
- View/download PDF
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