Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification

ABSTRACT Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.

Download Full-text

A metalloproteinase extracellularly released byCrithidia deanei

Canadian Journal of Microbiology ◽

10.1139/w03-081 ◽

2003 ◽

Vol 49 (10) ◽

pp. 625-632 ◽

Cited By ~ 15

Author(s):

Claudia Masini d'Avila-Levy ◽

Rodrigo F Souza ◽

Rosana C Gomes ◽

Alane B Vermelho ◽

Marta H Branquinha

Keyword(s):

Molecular Mass ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Residual Activity ◽

Major Protein ◽

Motile Cells ◽

Sds Page ◽

Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The incorporation of gelatin into SDSPAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Download Full-text

Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

Journal of Bacteriology ◽

10.1128/jb.181.1.91-99.1999 ◽

1999 ◽

Vol 181 (1) ◽

pp. 91-99 ◽

Cited By ~ 111

Author(s):

Hisayo Ono ◽

Kazuhisa Sawada ◽

Nonpanga Khunajakr ◽

Tao Tao ◽

Mihoko Yamamoto ◽

...

Keyword(s):

Molecular Mass ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Apparent Molecular Mass ◽

Optimum Temperature ◽

Sds Page ◽

Halomonas Elongata ◽

Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

Download Full-text

Isolation and purification of a Campylobacter upsaliensis autolysin

Canadian Journal of Microbiology ◽

10.1139/w98-125 ◽

1999 ◽

Vol 45 (1) ◽

pp. 23-30

Author(s):

Somchai Santiwatanakul ◽

Noel R Krieg

Keyword(s):

Escherichia Coli ◽

Molecular Mass ◽

Micrococcus Luteus ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Isolation And Purification ◽

Whole Cells ◽

Sds Page ◽

Autolytic Activity ◽

Campylobacter Upsaliensis

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS) - PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.Key words: autolysin, Campylobacter upsaliensis, zymogram, murein hydrolase.

Download Full-text

Glutamyl-tRNA Reductase of Chlorobium vibrioforme Is a Dissociable Homodimer That Contains One Tightly Bound Heme per Subunit

Journal of Bacteriology ◽

10.1128/jb.187.13.4444-4450.2005 ◽

2005 ◽

Vol 187 (13) ◽

pp. 4444-4450 ◽

Cited By ~ 24

Author(s):

Alaka Srivastava ◽

Samuel I. Beale

Keyword(s):

Molecular Mass ◽

Column Chromatography ◽

Aminolevulinic Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Affinity Column ◽

Chlorobium Vibrioforme ◽

Sds Page

ABSTRACT δ-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. To characterize the GTR protein, the hemA gene from C. vibrioforme was cloned into expression plasmids that added an N-terminal His6 tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5% (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His6-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNAGlu and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of pep tide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of β-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3- dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70% less bound heme than control GTR, and it was inhibited by hemin in vitro.

Download Full-text

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646313 ◽

1992 ◽

Vol 68 (05) ◽

pp. 534-538 ◽

Cited By ~ 5

Author(s):

Nobuhiko Yoshida ◽

Shingi Imaoka ◽

Hajime Hirata ◽

Michio Matsuda ◽

Shinji Asakura

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl Sulfate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Tetraacetic Acid ◽

Fibrin Monomer ◽

Sds Page ◽

Thrombotic Tendency ◽

Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Download Full-text

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

YARSI Medical Journal ◽

10.33476/jky.v26i2.394 ◽

2018 ◽

Vol 26 (2) ◽

pp. 058

Author(s):

Anna P. Roswiem ◽

Triayu Septiani

Keyword(s):

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Sds Page

Bahan baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.

Download Full-text

Majra Honey Abrogated the Normal and Cancer Cells Proliferation Inhibition by Juniperus procera Extract and Extract/Honey Generated AgNPs

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200213104224 ◽

2020 ◽

Vol 20 (8) ◽

pp. 970-981

Author(s):

Hamed A. Ghramh ◽

Essam H. Ibrahim ◽

Mona Kilnay

Keyword(s):

Anticancer Activity ◽

Cancer Cells ◽

Biological Activities ◽

Sugar Content ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Bioactive Molecules ◽

Sds Page ◽

Splenic Cells ◽

Juniperus Procera

Background: Juniperus procera and Majra honey are well-known as a folk medicine in many countries. Objectives: This work aimed to study the immunomodulatory effects after mixing Majra honey, J. procera water leaves extract and silver Nanoparticles (AgNPs) on immune or cancer cells. Methods: Juniperus procera water leaves extract and 20% Majra honey were prepared. Both the extract and honey were used separately to synthesize AgNPs. AgNPs were characterized using UV/Vis spectrophotometry and electron microscopy. Bioactive molecules in honey and the extract were explored using Fourier Transform Infrared (FT-IR) spectroscopy. Protein profile of honey was explored using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) and honey sugar content was determined using High- Performance Liquid Chromatography (HPLC). Biological activities of honey and the extract were tested. Results: The results demonstrated the ability of the extract/honey to produce AgNPs in a spherical shape. The extract/honey contained many functional groups. SDS-PAGE of Majra honey showed many protein bands. HPLC revealed honey is of good quality and no external additives are added to it. The extract and extract+ AgNPs inhibited the growth of normal rat splenic cells while honey stimulated it. The extract+honey turned stimulatory to the splenic cells’ growth and significantly diminished the inhibitory potential of the extract containing AgNPs. Both the extract and honey have antimicrobial activities, this potential increased in the presence of AgNPs. Honey and Honey+AgNPs inhibited HepG2 cancer cell proliferation while Hela cell growth inhibited only with honey+AgNPs. Conclusion: Both honey and the extract have antibacterial and immunomodulatory potentials as well as the power to produce AgNPs. Majra honey alone showed anticancer activity against HepGe2 cells, but not against Hela cells, and when contained AgNPs had anticancer activity on both cell lines. Mixing of Majra honey with J. procera extract showed characterized immunomodulatory potentials that can be described as immunostimulant.

Download Full-text

Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00125-2 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Preeti Anand ◽

Jay Prakash Pandey ◽

Dev Mani Pandey

Keyword(s):

San Francisco ◽

Tertiary Structure ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Evolutionary Genetics ◽

Imaging Analysis ◽

Silk Cocoon ◽

Natural Color ◽

Sds Page

Abstract Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

Download Full-text

Study on Degradation of Radix Isatidis Polypeptide in Artificial Gastrointestinal Environment

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.989-994.1020 ◽

2014 ◽

Vol 989-994 ◽

pp. 1020-1024

Author(s):

Nan Nan ◽

Xi Jing Liu

Keyword(s):

Gel Electrophoresis ◽

Free Amino ◽

High Performance ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Radix Isatidis ◽

Sds Page ◽

Electrophoresis Method ◽

Amino Acid Levels ◽

Different Molecular Weight

Radix Isatidis is a traditional Chinese medicine for treatment of influenza and inflammation in China. In this paper, in order to study the degradation situation of Radix Isatidis polypeptide in artificial gastrointestinal environment, the SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) method was used to detect the degradation of Radix Isatidis polypeptide in artificial intestinal juice and gastric juice, and it showed that Radix Isatidis peptides could be degradated to different degrees. HPLC (High Performance Liquid Chromatography) was used to determine the change of peptides degradation, and it indicated that free amino acid levels did not change significantly. The result after degradation was also detected by BCA method, and it showed that there were still a large number of polypeptides in the liquid. From this experiment we can come to this conclusion that Radix Isatidis polypeptides in artificial gastrointestinal juice mostly degraded into a series of different molecular weight peptides.

Download Full-text

Immunoreactivity of Lupine and Soybean Allergens in Foods as Affected by Thermal Processing

Foods ◽

10.3390/foods9030254 ◽

2020 ◽

Vol 9 (3) ◽

pp. 254 ◽

Cited By ~ 3

Author(s):

Caterina Villa ◽

Mónica B. M. V. Moura ◽

Joana Costa ◽

Isabel Mafra

Keyword(s):

Trypsin Inhibitor ◽

Thermal Processing ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Meat Products ◽

Soybean Trypsin Inhibitor ◽

Thermal Treatments ◽

Food Matrix ◽

Soybean Proteins ◽

Sds Page

Lupine and soybean are important technological aids for the food industry. However, they are also capable of inducing severe allergic reactions in food-sensitized/allergic individuals. In this context, this work intended to study the combined effects of thermal processing and food matrix on the immunoreactivity of lupine and soybean proteins used as ingredients in bakery and meat products, respectively. For this purpose, the effects of baking, mild oven cooking, and autoclaving on the protein profiles were evaluated, using model mixtures simulating the production of lupine-containing breads and soybean-containing cooked hams/sausages, by native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting using specific antibodies. The results showed that lupine gamma-conglutin immunoreactivity was slightly decreased in wheat flour mixtures compared to rice, but it was more pronounced in baked products. In meat mixtures, substantial protein fragmentation was noted after autoclaving, with decreased immunoreactivity of soybean trypsin inhibitor. The analysis of 22 commercial products enabled the identification of lupine gamma-conglutin in four bakery samples and soybean trypsin-inhibitor in five sausages, and further differentiated autoclaved from other milder thermally treated products. Generally, the immunoreactivity of target proteins was reduced by all the tested thermal treatments, though at a higher extent after autoclaving, being slightly altered by the food matrix.

Download Full-text