scholarly journals BACTERIAL AGGLUTINATION BY A LECTIN FROM THE LEAVES OF THE MEDICINAL PLANT, PIMENTA DIOICA (L.) MERR

2018 ◽  
Vol 10 (4) ◽  
pp. 35 ◽  
Author(s):  
Surya P H ◽  
Elyas K K ◽  
Deepti Madayi
Keyword(s):  
Escherichia Coli ◽  
Medicinal Plant ◽  
Biochemical Characterization ◽  
Exchange Chromatography ◽  
Bacterial Typing ◽  
Gram Negative ◽  
Sds Page ◽  
Bacterial Filters ◽  
Pimenta Dioica ◽  
Bacterial Agglutination

Objective: The current investigation involves the purification, characterization of the lectin from the leaves of Pimenta dioica (L.) Merr. (Myrtaceae) a medicinal plant, and its application in bacterial typing.Methods: A lectin was purified from the leaves by cation exchange chromatography. SDS PAGE revealed the molecular weight of the purified lectin. Biochemical characterization was carried out by performing various tests. Hemagglutination inhibition was conducted to detect the sugar specificity. Additionally, bacterial agglutination was performed to predict whether the purified lectin was able to agglutinate the bacterial strains.Results: SDS PAGE analysis revealed the lectin to be a tetramer in the range of 43-66 kDa. The purified lectin agglutinated human, avian, and mouse erythrocytes, and was inhibited by 125 mmol of mannose and xylose. The lectin was stable at 0-60 ° C for 30 min and was unaffected by either 2-Mercaptoethanol (2-ME) or Dithiothreitol (DTT) (50-250µM). A pH of 6.0–8.0 was found optimum for its activity and was nearly independent of metal ions. The purified lectin contained about 20% carbohydrate as estimated by Anthrone method. Purified lectin agglutinated the Gram-negative Escherichia coli and Proteus vulgaris.Conclusion: The isolated lectin was found to possess significant hemagglutinating activity. Due to its ability to agglutinate Gram negative bacteria such as Escherichia coli and Proteus vulgaris, it could be used for bacterial typing and for the design of bacterial filters.

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 Biochemical Characterization of a Thiol-Activated, Oxidation Stable Keratinase from Bacillus pumilus KS12

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Rinky Rajput ◽  
Richa Sharma ◽  
Rani Gupta
Keyword(s):  
Ion Exchange ◽  
Bacillus Pumilus ◽  
Biochemical Characterization ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amidolytic Activity ◽  
Alkaline Serine Protease ◽  
Sds Page ◽  
Temperature Optima

An extracellular keratinase from Bacillus pumilus KS12 was purified by DEAE ion exchange chromatography. It was a 45 kDa monomer as determined by SDS PAGE analysis. It was found to be an alkaline, serine protease with pH and temperature optima of 10 and 60C, respectively. It was thiol activated with two- and eight-fold enhancement in presence of 10 mM DTT and β-mercaptoethanol, respectively. In addition, its activity was stimulated in the presence of various surfactants, detergents, and oxidizing agents where a nearly 2- to 3-fold enhancement was observed in presence of H2O2 and NaHClO3. It hydrolyzed broad range of complex substrates including feather keratin, haemoglobin, fibrin, casein,and α-keratin. Analysis of amidolytic activity revealed that it efficiently cleaved phenylalanine → leucine → alanine- p-nitroanilides. It also cleaved insulin B chain between Val2- Asn3, Leu6-Cys7 and His10-Leu11 residues.

A fraction from Escherichia coli with anti-Aspergillus properties

2005 ◽  
Vol 54 (4) ◽  
pp. 375-379 ◽  
Author(s):  
V Yadav ◽  
R Mandhan ◽  
Rajesh Dabur ◽  
A K Chhillar ◽  
J Gupta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Concentration ◽  
Exchange Chromatography ◽  
Active Molecule ◽  
Standard Drug ◽  
E Coli ◽  
Variable Activity ◽  
Sds Page ◽  
Haemolytic Assay

The products of various strains of Escherichia coli (BL21, DH5α, HB101 and XL Blue) were investigated for antimycotic properties using pathogenic isolates of Aspergillus. Co-culture experiments revealed that E. coli strains exhibited variable activity against Aspergillus fumigatus. The lysates prepared from DH5α, HB101 and XL Blue strains of E. coli showed inhibitory activity against A. fumigatus in the protein concentration range of 62.50 to 250.00 μg ml−1. The highest activity was seen in the lysate of BL21, which inhibited the growth of A. fumigatus and Aspergillus flavus completely at a concentration of 31.25 μg protein ml−1. The MIC of BL21 lysate against Aspergillus niger was found to be 62.50 μg ml−1. The in vitro toxicity of BL21 lysate was evaluated using a haemolytic assay. A BL21 lysate protein concentration of 1250.00 μg ml−1 was found to be nontoxic to human erythrocytes. The standard drug amphotericin B lysed 100 % of erythrocytes at a concentration of 37.50 μg ml−1. SDS-PAGE showed the presence of at least 15 major proteins in the lysate of BL21. Ion-exchange chromatography resolved the BL21 lysate into five fractions and fraction III was found to be endowed with anti-Aspergillus properties. The MIC of this fraction was found to be 3.90 μg ml−1. Further work on the purification of the active molecule and its characterization is in progress.

scholarly journals Identification and Biochemical Characterization of a Novel Protein Phosphatase 2C-Like Ser/Thr Phosphatase in Escherichia coli

2018 ◽  
Vol 200 (18) ◽  
Author(s):  
Krithika Rajagopalan ◽  
Elizabeth Nagle ◽  
Jonathan Dworkin
Keyword(s):  
Escherichia Coli ◽  
Protein Phosphatase ◽  
Biochemical Characterization ◽  
Regulatory Protein ◽  
Negative Bacterium ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Novel Protein

Regulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria, including the model Gram-negative bacteriumEscherichia coli, demonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thr phosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

scholarly journals PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF A T/TN SPECIFIC LECTIN FROM LEPECHINIA BULLATA SEEDS (LAMIACEAE)

2017 ◽  
Vol 9 (10) ◽  
pp. 165
Author(s):  
Wilches Torres A. ◽  
Rojas Caraballo J. ◽  
Sanabria E. ◽  
Reyes MontaÑo E ◽  
FernÁndez Alonso Jl ◽  
...  
Keyword(s):  
Isoelectric Focusing ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reducing Conditions ◽  
Sds Page ◽  
Unique Band

Objective: This study focused on purifying and characterizing a lectin from Lepechinia bullata (L. bullata) seeds, and determining its specificity towards tumour-associated carbohydrate-antigens.Methods: Pigments were removed by washing the seeds with NH4OH 0.1 M pH 9.4 and treating the crude extracts with Pectinex®. The purification procedure consisted of anion exchange chromatography on diethylaminoethyl (DEAE)-Sephadex followed by affinity chromatography. For the characterization, the phase was used polyacrylamide gel electrophoresis-sodium dodecyl sulphate (SDS-PAGE), isoelectric focusing, hemagglutination assays, enzyme-linked lectinosorbent assay (ELLA) and thermal shift assay (TSA).Results: 6.2 mg of lectin were obtained from 100 g of seeds. It was able to agglutinate enzymatically treated erythrocytes with a minimal required lectin concentration of 7 μg. ml-1. Strong binding to asialo bovine submaxillary mucine (aBSM) was determined, corroborating Tn recognition.The isoelectric focusing showed a unique band at pH 8.5. Lectin pure shown bands at 28, 48 and 93 kDa by SDS-PAGE, with an incomplete dissociation of the last species despite trying several reduction conditions. By preparative electrophoresis under different conditions, three species were observed too, in all fractions one band at 28 kDa on Tricine-PAGE in reducing and no reducing conditions were found.Amino acid composition, carbohydrate content, thermal stability and Ca2+and Mn2+requirements were determined. N-acetylgalactosamine (GalNAc) and desialylated mucins inhibited the agglutinant activity on human cells. Fetuin inhibited hemagglutination of rabbit erythrocytes.Conclusion: A new lectin was isolated and characterized from L. bullata seeds, it recognizes T/Tn antigen and shows some similarities with other Lamiaceae lectins.

Protein profiles of Escherichia coli and Staphylococcus warneri are altered by photosensitization with cationic porphyrins

2015 ◽  
Vol 14 (6) ◽  
pp. 1169-1178 ◽  
Author(s):  
Eliana Alves ◽  
Ana Cristina Esteves ◽  
António Correia ◽  
Ângela Cunha ◽  
Maria A. F. Faustino ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Degradation ◽  
Protein Profiles ◽  
Low Light ◽  
Gram Negative ◽  
Cationic Porphyrins ◽  
Staphylococcus Warneri ◽  
Gram Positive Bacteria ◽  
Bacterial Proteins ◽  
Sds Page

Photosensitization of Gram(−)negative and Gram(+)-positive bacteria with cationic porphyrins induces overall protein degradation, revealed by SDS-PAGE, the tricationic one being extremely effective in degrading most bacterial proteins with low light doses.

scholarly journals Cloning, periplasmic expression and purification of hGM-CSF (Human Granulocyte - Macrophage ColonyStimulating Factor) in Escherichia coli

2014 ◽  
Vol 17 (4) ◽  
pp. 12-19
Author(s):  
Phu Sang Nguyen ◽  
Thanh Thao Nguyen ◽  
Hieu Tran Van
Keyword(s):  
Escherichia Coli ◽  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cloning And Expression ◽  
Peptide Sequence ◽  
Periplasmic Fraction ◽  
E Coli ◽  
Gm Csf ◽  
Sds Page

Human GM-CSF is a cytokine consisting of 127 amino acid residues, with four cysteines being involved in two disulfide bonds. Although GM-CSF is glycosylated in its natural form, the glycosylation perhaps has not been involved in its biological function. GM-CSF stimulates the survival, proliferation, and differentiation of hem at opoietic progenitor cells and also enhances the functional properties of mature myeloid cells. GM-CSF is used as a therapeutic agent in various clinical cases such as neutropenia following chemotherapy, bone marrow transplantation, acute myeloid leukemia… In this study, we report the results on the cloning and expression of recombinant human GM-CSF in the periplasmic space of Escherichia coli. The hGM-CSF gene was amplified by polymerase chain reaction using two oligonucleotide primers containing NcoI and XhoI restriction sites. This DNA fragment was successfully cloned between the NcoI and Xho I sites of the plasmid pET-22b, in frame with the pelB signal peptide sequence. The expression vector pET-hGM was transformed into E. coli BL21(DE3) and the transformants were induced by IPTG and examined for hGM-CSF production. Periplasmic proteins were released by osmotic shock treatment. The expression of recombinant hGM-CSF was evaluated by SDS–PAGE in total, cytoplasmic and periplasmic fractions. The recombinant hGMCSF in periplasmic fraction was then subjected to ion exchange chromatography using Q Sepharose FF column with saltincrement elution step. SDS-PAGE showed there was a visible expression of recombinant hGM-CSF in the periplasmic fraction of the E. coli BL21(DE3)/pET-hGM and a purified band with the purity of 97.4% after ion exchange chromatography. This result was further confirmed by Western blot using anti-hGM-CSF antibody.

scholarly journals Biochemical Characterization of the THIN-B Metallo-β-Lactamase of Janthinobacterium lividum

2004 ◽  
Vol 48 (12) ◽  
pp. 4778-4783 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Teresa Lopizzo ◽  
Sabrina Liberatori ◽  
Manuela Prenna ◽  
Maria Cristina Thaller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ternary Complex ◽  
Biochemical Analysis ◽  
Biochemical Characterization ◽  
Expression System ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Janthinobacterium Lividum ◽  
Individual Rate

ABSTRACT The THIN-B metallo-β-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum, was overproduced in Escherichia coli by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa. It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator. Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex.

Antimicrobial and cytotoxic activity of Macrophomina phaseolina isolated from gummosis infected citrus reticulata

2013 ◽  
pp. 125-133
Author(s):  
Rubal C Das ◽  
Rajib Banik ◽  
Robiul Hasan Bhuiyan ◽  
Md Golam Kabir
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Brine Shrimp ◽  
Inhibitory Concentration ◽  
Lethal Dose ◽  
Macrophomina Phaseolina ◽  
Chloroform Extract ◽  
Citrus Reticulata ◽  
Gram Negative Bacteria ◽  
Gram Negative

Macrophomina phaseolina is one of the pathogenic organisms of gummosis disease of orange tree (Citrus reticulata). The pathogen was identified from the observation of their colony size, shape, colour, mycelium, conidiophore, conidia, hyaline, spore, and appressoria in the PDA culture. The crude chloroform extracts from the organism showed antibacterial activity against a number of Gram positive and Gram-negative bacteria. The crude chloroform extract also showed promising antifungal activity against three species of the genus Aspergillus. The minimum inhibitory concentration (MIC) of the crude chloroform extract from M. phaseolina against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Shigella sonnie were 128 ?gm, 256 ?gm, 128 ?gm and 64 ?gm/ml respectively. The LD50 (lethal dose) values of the cytotoxicity assay over brine shrimp of the crude chloroform extract from M. phaseolina was found to be 51.79 ?gm/ml. DOI: http://dx.doi.org/10.3329/cujbs.v5i1.13378 The Chittagong Univ. J. B. Sci.,Vol. 5(1 &2):125-133, 2010

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