scholarly journals Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975

1999 ◽  
Vol 341 (2) ◽  
pp. 285-291 ◽  
Author(s):  
Donna D. SONG ◽  
Nicholas A. JACQUES
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Streptococcus Salivarius ◽  
Acetobacter Diazotrophicus ◽  
E Coli ◽  
Ping Pong ◽  
Mechanism Of Catalysis ◽  
Enzyme Intermediate

The recombinant fructosyltransferase (Ftf) of Streptococcus salivarius was expressed in Escherichia coli and purified to electrophoretic homogeneity after a combination of adsorption, ion-exchange and gel-filtration chromatography. The N-terminal signal sequence of the Ftf was removed by E. coli at the same site as in its natural host. The purified Ftf exhibited maximum activity at pH 6.0 and 37 °C, was activated by Ca2+, but inhibited by the metal ions Cu2+, Zn2+, Hg2+ and Fe3+. The enzyme catalysed the transfer of the fructosyl moiety of sucrose to a number of acceptors, including water, glucose and sucrose via a Ping Pong mechanism involving a fructosyl-enzyme intermediate. While this mechanism of catalysis is utilized by the levansucrases of Bacillus subtilis and Acetobacter diazotrophicus and the values of the kinetic constants for the three enzymes are similar, sucrose was a far more efficient fructosyl-acceptor for the Ftf of S. salivarius than for the two other enzymes.

scholarly journals Purification and characterization of [acyl-carrier-protein] acetyltransferase from Escherichia coli

1988 ◽  
Vol 250 (3) ◽  
pp. 789-796 ◽  
Author(s):  
P N Lowe ◽  
S Rhodes
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
E Coli ◽  
Step Procedure ◽  
Ping Pong ◽  
Protein Acetyltransferase ◽  
Enzyme Intermediate ◽  
Specific Reagent

A multi-step procedure has been developed for the purification of [acyl-carrier-protein] acetyltransferase from Escherichia coli, which allows the production of small amounts of homogeneous enzyme. The subunit Mr was estimated to be 29,000 and the native Mr was estimated to be 61,000, suggesting a homodimeric structure. The catalytic properties of the enzyme are consistent with a Bi Bi Ping Pong mechanism and the existence of an acetyl-enzyme intermediate in the catalytic cycle. The enzyme was inhibited by N-ethylmaleimide and more slowly by iodoacetamide in reactions protected by the substrate, acetyl-CoA. However, the enzyme was apparently only weakly inhibited by the thiol-specific reagent methyl methanethiosulphonate. The nature of the acetyl-enzyme intermediate is discussed in relationship to that found in other similar enzymes from E. coli, yeast and vertebrates.

scholarly journals Characterization and in Vivo Cloning of prlC, a Suppressor of Signal Sequence Mutations in Escherichia coli K12

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 513-521
Author(s):  
Nancy J Trun ◽  
Thomas J Silhavy
Keyword(s):  
Escherichia Coli ◽  
Genetic Mapping ◽  
Signal Sequence ◽  
Illegitimate Recombination ◽  
Mutant Phenotype ◽  
E Coli ◽  
Physical Location ◽  
Sequence Deletion ◽  
New Alleles

ABSTRACT The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamBsignal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using λplacMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

scholarly journals Overproduction of Penicillin-Binding Protein 2 and Its Inactive Variants Causes Morphological Changes and Lysis in Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 4975-4983 ◽  
Author(s):  
Blaine A. Legaree ◽  
Calvin B. Adams ◽  
Anthony J. Clarke
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Morphological Changes ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Prolonged Incubation ◽  
Lytic Transglycosylases ◽  
E Coli ◽  
Derivatives Of

ABSTRACT Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.

scholarly journals Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain)

1973 ◽  
Vol 131 (4) ◽  
pp. 833-841 ◽  
Author(s):  
Donald Stribling ◽  
Richard N. Perham
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Metal Ions ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Class Ii ◽  
Class I ◽  
E Coli ◽  
Km Value ◽  
Bivalent Metal Ions

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca2+, Zn2+. It is a dimer with a molecular weight of approx. 70000, and the Km value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH4 in the presence of substrate. The Km value for fructose diphosphate is about 20μm (although the Lineweaver–Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.

scholarly journals Gonococcal MinD Affects Cell Division inNeisseria gonorrhoeae and Escherichia coli and Exhibits a Novel Self-Interaction

2001 ◽  
Vol 183 (21) ◽  
pp. 6253-6264 ◽  
Author(s):  
Jason Szeto ◽  
Sandra Ramirez-Arcos ◽  
Claude Raymond ◽  
Leslie D. Hicks ◽  
Cyril M. Kay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Morphological Changes ◽  
Shuttle Vector ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
E Coli ◽  
Two Hybrid

ABSTRACT The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this operon. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from N. gonorrhoeae(MinDNg) in this mutant. Hence, MinDNg is required for maintaining proper cell division and growth in N. gonorrhoeae. Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinDNg is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinCNg and MinDNg from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinCNg or MinDNg alone did not display noticeable morphological changes. These studies suggest that MinDNg is involved in inhibiting gonococcal cell division, likely in conjunction with MinCNg. The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinDNg in wild-type Escherichia coli led to cell filamentation, while overexpression in an E. coli minD mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinDNg revealed a novel MinDNgself-interaction. We have also shown by yeast two-hybrid analysis that MinD from E. coli interacts with itself and with MinDNg. These results indicate that MinDNg is required for maintaining proper cell division and growth in N. gonorrhoeae and suggests that the self-interaction of MinD may be important for cell division site selection across species.

The purification from Escherichia coli of a protein relaxing superhelical DNA

1976 ◽  
Vol 54 (4) ◽  
pp. 301-306 ◽  
Author(s):  
M. G. Burrington ◽  
A. R. Morgan
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Rapid Screening ◽  
Intact Protein ◽  
Winding Number ◽  
Circular Dna ◽  
E Coli ◽  
The Individual ◽  
Negative Supercoils

The Escherichia coli omega protein was first described by Wang (Wang, J.C.: J. Mol. Biol. 55, 523–533 (1971)) as having the ability to relax supercoiled covalently-closed circular DNA by changing the topological winding number, α. We have developed a rapid assay for omega activity which has allowed us to purify the protein to homogeneity. It appears to be an αβ-ype subunit protein with a molecular weight of the intact protein of about 80 000 (determined by gel filtration) and of the individual subunits of 56 000 and 31 000 (sodium dodecyl sulfate polyacrylamide gels). We have confirmed Wang's observation that it only partly relaxes negative supercoils, and is not active on positive supercoils. Its characteristics with respect to pH, salts, temperature and chromatography are described. A method for rapid screening of E. coli for omega mutants is described.

scholarly journals Study of Antibacterial Activity of Chenopodium album Leaves Extract

2018 ◽  
Vol 10 (01) ◽  
Author(s):  
Kaur M. ◽  
Sharma S. ◽  
Garg S. ◽  
Arora M.
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Bacillus Subtilis ◽  
Chenopodium Album ◽  
Antibacterial Activities ◽  
Maximum Activity ◽  
Zone Of Inhibition ◽  
Plate Method ◽  
E Coli ◽  
Test Organisms

This study describes the antibacterial activities of three different solvent extracts of leaves of Chenopodium album. Methanol, acetone and chloroform extracts of C. album were prepared. The antibacterial activity was assessed using well plate method and were examined for the size of zone of inhibition. Different extracts were investigated against the test organisms namely Lactobacillus, Bacillus subtilis and Escherichia coli. The maximum activity was observed at 100% concentration of different extracts of leaves. The maximum zone of inhibition for 100% concentration were observed as E. coli (19 mm) and Lactobacillus (19 mm) in diameter respectively. C. album did not show any antibacterial activity against B. subtilis. Antibacterial activity was compared with standard Amoxicillin and it was found to be 23 mm diameter for Lactobacillus and 25 mm for both E. coli and B. subtilis in terms of zone of inhibition.

Implementation of a novel self-induced promoter for the expression of pharmaceutical peptides in Escherichia coli: YY(3-36) peptide

2019 ◽  
Vol 0 (0) ◽  
Author(s):  
Amir Hossein Momen ◽  
Naser Harzandi ◽  
Azam Haddadi ◽  
Bijan Bambai
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Peptide Yy ◽  
Signal Sequence ◽  
Expression System ◽  
Promoter Sequence ◽  
Scale Production ◽  
Signal Sequences ◽  
T7 Promoter ◽  
E Coli

Abstract Background Increasing the expression rate of recombinant mammalian hormones in Escherichia coli by combining efficient promoters and signal sequences is a never ending process. A self-induced promoter will have some beneficial gains compared to the classical T7 promoter or its variants with isopropyl β-D-1-thiogalactopyranoside (IPTG) as the inducer. Obesity is the prime suspect in widespread frequency of diabetes type II and cardiovascular diseases worldwide. YY (tyrosine-tyrosine) peptide is a local acting hormone, controlling appetite. Excitingly, it was has been shown that a truncated version of the YY peptide, YY(3-36) peptide, has potential as a worthy biopharmaceutical agent in the fight against obesity. Materials and methods To develop an economical expression system for the large scale production of the peptide in Gram-negative bacteria, we introduced a promoter sequence upstream of a chimeric gene for the extracellular expression of this peptide with the assistance of a signal sequence of asparaginase II from E. coli. This system has the advantage of producing a complete sequence of a truncated YY peptide, YY(3-36), without any extra tags that would require further removal with the assistance of expensive specific proteases and reduced the downstream steps, significantly. Results Recombinant production of YY(3-36) peptide under a self-induced promoter proves the efficacy of the asparaginase II signal sequence as a communicator of foreign peptides and proteins into the extracellular space of E. coli. Conclusions The application of fusion protein expression of biopharmaceuticals, especially mammalian hormones, in prokaryotic systems with the help of native signal sequences makes some common tags with expensive proteases for the removal of the attached protein Tag redundant.

scholarly journals A Novel Pathogenicity Island Integrated Adjacent to the thrW tRNA Gene of Avian Pathogenic Escherichia coli Encodes a Vacuolating Autotransporter Toxin

2003 ◽  
Vol 71 (9) ◽  
pp. 5087-5096 ◽  
Author(s):  
V. R. Parreira ◽  
C. L. Gyles
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Trna Gene ◽  
Signal Sequence ◽  
Sequence Similarity ◽  
Pathogenicity Island ◽  
Open Reading Frames ◽  
Integrase Gene ◽  
E Coli ◽  
Reading Frames

ABSTRACT We report the complete nucleotide sequence and genetic organization of the Vat-encoding pathogenicity island (PAI) of avian pathogenic Escherichia coli strain Ec222. The 22,139-bp PAI is situated adjacent to the 3′ terminus of the thrW tRNA gene, has a G+C content of 41.2%, and includes a bacteriophage SfII integrase gene, mobile genetic elements, two open reading frames with products exhibiting sequence similarity to known proteins, and several other open reading frames of unknown function. The PAI encodes an autotransporter protein, Vat (vacuolating autotransporter toxin), which induces the formation of intracellular vacuoles resulting in cytotoxic effects similar to those caused by the VacA toxin from Helicobacter pylori. The predicted 148.3-kDa protein product possesses the three domains that are typical of serine protease autotransporters of Enterobacteriaceae: an N-terminal signal sequence of 55 amino acids, a 111.8-kDa passenger domain containing a modified serine protease site (ATSGSG), and a C-terminal outer membrane translocator of 30.5 kDa. Vat has 75% protein homology with the hemagglutinin Tsh, an autotransporter of avian pathogenic E. coli. A vat deletion mutant of Ec222 showed no virulence in respiratory and cellulitis infection models of disease in broiler chickens. We conclude that the newly described PAI and Vat may be involved in the pathogenicity of avian septicemic E. coli strain Ec222 and other avian pathogenic E. coli strains.

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