scholarly journals Purification and characterization of a diptericin homologue from Sarcophaga peregrina (flesh fly)

1992 ◽  
Vol 287 (2) ◽  
pp. 573-578 ◽  
Author(s):  
M Ishikawa ◽  
T Kubo ◽  
S Natori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Bactericidal Activity ◽  
Fat Body ◽  
Amino Acid Sequencing ◽  
Purification And Characterization ◽  
E Coli ◽  
Escherichia Coli Cells

A protein with a molecular mass of 8 kDa was found to be synthesized specifically when the fat-body from injured Sarcophaga peregrina larvae was cultured in vitro. This protein was purified from the haemolymph of the injured larvae to near-homogeneity. Partial amino acid sequencing revealed that this protein is a diptericin homologue. It showed bactericidal activity on growing, but not resting Escherichia coli cells. E. coli cells become elongated on treatment with this protein.

scholarly journals Characterization of human interleukin 2 derived from Escherichia coli

1985 ◽  
Vol 229 (2) ◽  
pp. 429-439 ◽  
Author(s):  
S M Liang ◽  
B Allet ◽  
K Rose ◽  
M Hirschi ◽  
C M Liang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Dna Sequence ◽  
Protein A ◽  
Interleukin 2 ◽  
Coding Region ◽  
Disulphide Bridge ◽  
E Coli ◽  
Escherichia Coli Cells

Interleukin 2 isolated from Escherichia coli cells expressing the human interleukin gene has been characterized. The observed properties of the protein have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural human interleukin 2. The purified E. coli-derived interleukin 2 is a monomeric protein of Mr 15 000 with a sedimentation velocity of 1.86S. The amino acid composition of the protein and isoelectric point (7.7) are consistent with that part of the translated DNA sequence of the gene corresponding to the mature protein. A single disulphide bridge was identified between Cys-58 and Cys-105. C.d. suggested that interleukin 2 is predominantly alpha-helical in secondary structure. The E. coli-derived protein differed from natural interleukin 2 in the presence of N-terminal methionine and also in the absence of a carbohydrate moiety. Removal of the coding region for the first three amino acids of the natural interleukin 2 protein sequence (Ala-Pro-Thr) by site-specific mutagenesis resulted in a protein with N-terminal serine. The possibility that the specificity of the E. coli ribosomal methionine aminopeptidase may not recognize the sequence NH2-Met-Xaa-Pro is discussed (where Xaa is any amino acid residue).

scholarly journals Purification and characterization of the class-II D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes' strain)

1978 ◽  
Vol 169 (3) ◽  
pp. 633-641 ◽  
Author(s):  
S A Baldwin ◽  
R N Perham ◽  
D Stribling
Keyword(s):  
Escherichia Coli ◽  
Common Ancestor ◽  
Class Ii ◽  
Purification And Characterization ◽  
Thiol Compounds ◽  
E Coli ◽  
Optimum Ph ◽  
Class Ii Aldolase ◽  
Fructose 1,6 Bisphosphate

A new form of the class-II D-fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) of Escherichia coli (Crookes' strain) was isolated from an extract of glycerol-grown bacteria. It has a higher molecular weight (approx. 80000)than previous preparations of the enzyme and closely resembles the typical class-II aldolase from yeast in size and amino acid composition. On the other hand, its kinetic behaviour is not typical of a class-II aldolase. The enzyme has no requirement for thiol compounds either for stability or activity, added K+ ions have no effect, and the optimum pH for the cleavage activity is unusually high. The class-II enzymes from the prokaryote E. coli and the eukaryote yeast show no immunological identity. However, the similarity of their structures suggests that they have evolved from a common ancestor.

scholarly journals Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

1996 ◽  
Vol 314 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Johanneke L. H. BUSCH ◽  
Jacques L. J. BRETON ◽  
Barry M. BARTLETT ◽  
Richard JAMES ◽  
E. Claude HATCHIKIAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnetic Circular Dichroism ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Expression In Escherichia Coli ◽  
Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

scholarly journals Purification and Characterization of AsES Protein

2013 ◽  
Vol 288 (20) ◽  
pp. 14098-14113 ◽  
Author(s):  
Nadia R. Chalfoun ◽  
Carlos F. Grellet-Bournonville ◽  
Martín G. Martínez-Zamora ◽  
Araceli Díaz-Perales ◽  
Atilio P. Castagnaro ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Foliar Spray ◽  
Amino Acid Sequences ◽  
Purification And Characterization ◽  
Degenerate Primers ◽  
Acremonium Strictum ◽  
Rapid Amplification

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

scholarly journals Isolation and characterization of Escherichia coli phage and enhance its bactericidal activity that collaborative with kanamycin sulfate

2020 ◽  
Author(s):  
Liming Jiang ◽  
Rui Zheng
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Bactericidal Activity ◽  
Therapeutic Potential ◽  
Health Concern ◽  
Low Concentration ◽  
E Coli ◽  
Kanamycin Sulfate ◽  
Isolation And Characterization

Abstract Background: Escherichia coli is the most important and widespread bacteria in worldwide, which mainly found in contaminated food, human and animal faeces. Unfortunately, Some of E. coli strains are multidrug-resistant (MDR) pathogen leading significant public health concern globally. Biofilm is a multicellular community of microorganisms. Phages and their derivatives are ideal candidates for replacing or compensating for antibiotic problems in the future. Method: Here, we aimed to isolation and characterization of Escherichia coli phage and research its bactericidal activity that individually or collaborative with kanamycin sulfateResults: In this study, three virulent phages Flora, T4 and WJ were isolated from the laboratory and drug sample in Wuxi, China. It’s belonged to the Myoviridae family and optimum temperature is 42 ℃, optimum pH= 7, optimum MOI is 0.0001 and the genome size of Flora, T4 and WJ were 168, 909, 168903 and 168, 900 bp respectively. Flora has two exonuclease, whereas T4 and WJ have only one. Antibiotics have better bactericidal activity than phages in a low concentration medium of bacteria, nonetheless, phages have better bactericidal activity than antibiotics in a high concentration of bacteria, and that, collaboration of phages and antibiotics have better bactericidal activity effect than alone of phages or antibiotics in a low concentration medium of bacteria. Conclusion: The excellent performance of phage Flora for its therapeutic potential on clinic. The data of this study provided the strong evidence that the application of phage could reduce the growth and biofilm of E. coli that are important to maintain public health. Keywords: Escherichia coli, phage, lytic spectrum, biofilm, antibiotic

scholarly journals ISOLATION AND CHARACTERIZATION OF dnaX AND dnaY TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI

Genetics ◽  
1979 ◽  
Vol 92 (4) ◽  
pp. 1041-1059
Author(s):  
Joan M Henson ◽  
Herman Chu ◽  
Carleen A Irwin ◽  
James R Walker
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
E Coli ◽  
Temperature Sensitive Mutants ◽  
Isolation And Characterization ◽  
Ts Mutations

ABSTRACT Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated. Based on transduction by phage PI, dnaX and Y have been mapped at minutes 10.4-10.5 and 12.1, respectively, in the sequence dnaX purE dnaY. Both dnaXts36 and YtslO are recessive to wild-type alleles present on episomes. F13 carries both dnaX  + and Y  +; the shorter F210 carries dnaY  +, but not X  +. Lambda transducing phages that carry dnaX  + or Y  + have been isolated, and hybrid plasmids of Col E1 and E. coli DNA from the CLARKE and CARBON (1976) collection also carry portions of the dnaX purE dnaY region. Results obtained with the λ transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5.—The dnaXts36 mutant, after a shift to 42°, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold. When this mutant was shifted to M°, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount. Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42° and was partially in-hibited even at 30°.—When the dnaYtslO mutant was shifted to 42°, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold. At 44°, residual DNA synthesis amounted to a two-fold increase. Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42° or by "preincubation" of cells at 42° before toluene treatment.— The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded.

scholarly journals Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ

2010 ◽  
Vol 190 (4) ◽  
pp. 613-621 ◽  
Author(s):  
Julio O. Ortiz ◽  
Florian Brandt ◽  
Valério R.F. Matias ◽  
Lau Sennels ◽  
Juri Rappsilber ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Spatial Organization ◽  
Three Dimensional ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Three Dimensional Configuration

Ribosomes arranged in pairs (100S) have been related with nutritional stress response and are believed to represent a “hibernation state.” Several proteins have been identified that are associated with 100S ribosomes but their spatial organization has hitherto not been characterized. We have used cryoelectron tomography to reveal the three-dimensional configuration of 100S ribosomes isolated from starved Escherichia coli cells and we have described their mode of interaction. In situ studies with intact E. coli cells allowed us to demonstrate that 100S ribosomes do exist in vivo and represent an easily reversible state of quiescence; they readily vanish when the growth medium is replenished.

scholarly journals Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo

2005 ◽  
Vol 389 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Rajesh K. Soni ◽  
Parul Mehra ◽  
Gauranga Mukhopadhyay ◽  
Suman Kumar Dhar
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
E Coli ◽  
Dnab Helicase ◽  
H Pylori

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli

2015 ◽  
Vol 25 (6) ◽  
pp. 394-402 ◽  
Author(s):  
Taylor L. Fischer ◽  
Robert J. White ◽  
Katherine F.K. Mares ◽  
Devin E. Molnau ◽  
Justin J. Donato
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Wild Type ◽  
E Coli ◽  
Enoyl Acp Reductase ◽  
Selection For

<b><i>Background/Aims:</i></b> We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in <i>Escherichia coli</i>. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. <b><i>Methods:</i></b> ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into <i>E. coli</i>, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. <b><i>Results:</i></b> Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into <i>E. coli</i>, whereas the mutant remained susceptible to triclosan<i>. </i>Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. <b><i>Conclusion:</i></b> ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.

PURIFICATION AND CHARACTERIZATION OF DONKEY CHORIONIC GONADOTROPHIN

1980 ◽  
Vol 85 (3) ◽  
pp. 449-455 ◽  
Author(s):  
B. B. AGGARWAL ◽  
SUSAN W. FARMER ◽  
HAROLD PAPKOFF ◽  
FRANCESCA STEWART ◽  
W. R. ALLEN
Keyword(s):  
Amino Acid ◽  
Leydig Cell ◽  
Seminiferous Tubule ◽  
Chorionic Gonadotrophin ◽  
Purification And Characterization ◽  
Rat Testis ◽  
Amino Terminal ◽  
Biological Tests

Serum of the pregnant donkey, like that of the mare, contains a gonadotrophin of chorionic origin. The chorionic gonadotrophin of the donkey (dCG) has been isolated in purified form from the serum of pregnant donkeys using methodology previously employed for the purification of pregnant mare chorionic gonadotrophin (eCG). Unlike eCG, dCG is predominantly an LH in biological tests. In the in-vitro rat Leydig cell assay, dCG was as active as eCG, but in the in-vitro rat seminiferous tubule assay for FSH and in the augmentation assay, dCG was considerably less potent than eCG (1–10%). Specific rat testis radioreceptor assays for LH and FSH also showed dCG to be at least nine times more potent in LH than in FSH activity. Chemically, dCG was found to be similar to eCG in fractionation behaviour and glycoprotein nature. However, dCG had significantly less carbohydrate (31%) than had eCG (45%) and several differences were noted in a comparison of amino-acid compositions. A single amino-terminal residue, phenylalanine, was detected in dCG. Immunologically, dCG cross-reacted in homologous radioimmunoassays for eCG, equine LH and equine FSH, but its inhibition curves were all non-parallel with those of the respective equine gonadotrophin standards.

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