Isolation and purification of granule-associated proteins relevant for poly(3-hydroxybutyric acid) biosynthesis from methylotrophic bacteria relying on the serine pathway

1995 ◽  
Vol 41 (13) ◽  
pp. 124-130 ◽  
Author(s):  
C. G. Föllner ◽  
W. Babel ◽  
A. Steinbüchel
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Methylotrophic Bacteria ◽  
Hydroxybutyric Acid ◽  
Isolation And Purification ◽  
Small Proteins ◽  
Associated Proteins ◽  
Serine Pathway ◽  
Methylobacterium Rhodesianum ◽  
Leaky Mutants

The poly(3-hydroxybutyric acid) (PHB) granules from eight methylotrophic bacteria that use the serine pathway were isolated in a sucrose gradient (1–2 M); these bacteria included members of the genus Methylobacterium, Mycoplana rubra, and PHB-leaky mutants of Methylobacterium rhodesianum. As shown by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, the granules from all investigated methylotrophic strains revealed two major bands representing small proteins. An efficient purification procedure for these two low molecular weight proteins associated with the PHB granules was developed by solubilization of the proteins with Triton X-114 and affinity chromatography on Procion Blue-H-ERD.Key words: poly(3-hydroxybutyric acid), granule-associated proteins, methylotrophic bacteria.

Isolation and purification of a Campylobacter upsaliensis autolysin

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.

scholarly journals Amelogenins. Purification and partial characterization of proteins from developing bovine dental enamel

1973 ◽  
Vol 131 (3) ◽  
pp. 471-484 ◽  
Author(s):  
F. Michael Eggert ◽  
Grania A. Allen ◽  
Ralph C. Burgess
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dental Enamel ◽  
Polyacrylamide Gel ◽  
Sedimentation Equilibrium ◽  
Amino Acid Compositions ◽  
Small Proteins ◽  
Equilibrium Ultracentrifugation

1. Procedures are described for the purification of amelogenin electrophoretic components and their analysis for homogeneity by polyacrylamide-gel electrophoresis at both acidic and alkaline pH values. 2. Most of these components belonged to two main groups, termed the J group and the C group after their major electrophoretic components. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis indicated that, within each group, proteins were of similar size, but the C-group proteins were larger than those of the J group. 3. By sedimentation-equilibrium ultracentrifugation and amino acid analysis, the four J-group components were found to be very small proteins (mol. wt. 5500–3000) and, except for one, similar in amino acid composition. The components of the C group were found to be proteins of moderate size (mol. wt. 16800–16100) with very similar amino acid compositions. A third minor amelogenin group of intermediate size was also found, but not further analysed. Details of the results of the ultracentrifuge studies are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50014 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. 4. Two of the J-group components were similar to amelogenins isolated by other workers. 5. All amelogenins analysed were rich in proline, glutamic acid, histidine and methionine, and contained no half-cystine. Their amino acid compositions, combined with their molecular weights, serve to distinguish the amelogenins from both collagens and keratins.

HSP90 expression correlation with the freezing resistance of bull sperm

Zygote ◽  
2013 ◽  
Vol 22 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Peng Wang ◽  
Yan-Feng Wang ◽  
Hong Wang ◽  
Chun-Wei Wang ◽  
Lin-Sen Zan ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Sperm Quality ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Freezing Resistance ◽  
Hsp90 Expression ◽  
Sds Page ◽  
Associated Proteins ◽  
Holstein Bulls ◽  
Bull Sperm

SummaryTo date, there has been little improvement in cryopreservation of bull sperm due to lack of understanding of the freezing mechanisms. Therefore, this study set out to investigate expression levels of fertility-associated proteins in bull sperm, and in particular the relationship between the 90 kDa heat-shock protein (HSP90) and the sperm characteristics after freezing–thawing. Semen was collected from eight Holstein bulls by artificial vagina. Characteristics of these fresh semen, including sperm motility, morphology, viability and concentration, were evaluated. Sperm quality was also assessed after freezing–thawing. Eight ejaculates were divided into two groups based on freezing resistance and sperm motility. Sperm proteins were extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and western blotting were performed. SDS-PAGE results showed that there was substantial diversity in 90 kDa proteins in the frozen–thawed sperm and HSP90 was confirmed as one of the 90 kDa proteins by western blot. This study indicated that HSP90 expression correlated positively with sperm quality. The amount of expressed 90 kDa proteins in the high freezing resistance (HFR) group was significantly higher than that in the low freezing resistance (LFR) group (P< 0.05). Thus, higher expression of HSP90 could probably lead to the higher motility and freezing resistance of sperm found after freezing–thawing. Therefore, we concluded that level of HSP90 expression could be used to predict reliably and simply the freezing resistance of bull sperm.

scholarly journals Influence of the Poly-3-Hydroxybutyrate (PHB) Granule-Associated Proteins (PhaP1 and PhaP2) on PHB Accumulation and Symbiotic Nitrogen Fixation in Sinorhizobium meliloti Rm1021

2007 ◽  
Vol 189 (24) ◽  
pp. 9050-9056 ◽  
Author(s):  
Chunxia Wang ◽  
Xiaoyan Sheng ◽  
Raymie C. Equi ◽  
Maria A. Trainer ◽  
Trevor C. Charles ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Sinorhizobium Meliloti ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dry Weight ◽  
Granule Formation ◽  
Maldi Tof ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity ◽  
Associated Proteins

ABSTRACT Sinorhizobium meliloti cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in S. meliloti have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (phaP1) and SMc02111 (phaP2) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels. Medicago truncatula plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.

Isolation and Purification of Colicin ECL 12, a Bacteriocin That Inhibits Strains of Escherichia coli O157:H7†

1997 ◽  
Vol 60 (12) ◽  
pp. 1520-1528 ◽  
Author(s):  
WANDA J. LYON ◽  
DENNIS G. OLSON
Keyword(s):  
Escherichia Coli ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Viable Cell ◽  
Exchange Chromatography ◽  
Isolation And Purification ◽  
E Coli ◽  
Log Reduction ◽  
Cell Counts

A swine fecal isolate, identified as Escherichia coli ECL12, was found to produce an antimicrobial substance designated as colicin ECL12. Colicin ECL12 was inhibitory against 20 strains of E. coli O157:H7 previously isolated from both human and bovine feces. Identification of the producer strain was determined phenotypically by biochemical and morphological tests. Colicin ECL12 was sensitive to several proteolytic enzymes. Adsorption of colicin ECL12 to sensitive cells of E. coli O157:H7 was bactericidal, resulting in a 2 log reduction in viable cell counts. Colicin ECL12 was purified from strain ECL12 by cell extraction and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of colicin ECL12 resolved a single protein with a molecular weight of approximately 65,000.

Accumulation of poly(3-hydroxybutyric acid) and overproduction of exopolysaccharides in a mutant of a methylotrophic bacterium

1995 ◽  
Vol 41 (13) ◽  
pp. 55-59 ◽  
Author(s):  
Uta Breuer ◽  
Jörg-Uwe Ackermann ◽  
Wolfgang Babel
Keyword(s):  
Ammonium Phosphate ◽  
Sole Source ◽  
Chemical Mutagenesis ◽  
Methylotrophic Bacterium ◽  
Wild Type ◽  
Hydroxybutyric Acid ◽  
Serine Pathway ◽  
Methylobacterium Rhodesianum

The pink-pigmented facultatively methylotrophic bacterium Methylobacterium rhodesianum MB 126 is able to grow on methanol as the sole source of carbon and energy. Under certain conditions, e.g., limitation of ammonium, phosphate, or oxygen, carbon from methanol is channeled into poly(3-hydroxybutyric acid) (PHB) whereas other polymers or metabolites are hardly overproduced. A mutant of this strain, which we isolated after chemical mutagenesis, is impaired in its ability to synthesize PHB. Under the conditions mentioned above, the mutant still accumulated PHB, but in the absence of ammonium it simultaneously synthesized PHB and a considerable amount of an exopolysaccharide. This phenomenon was surprising insofar as the wild type did not produce exopolysaccharide in such amounts. An attempt was made to elucidate and discuss the possible reasons for these findings.Key words: methylotrophy, serine pathway bacteria, PHB, exopolysaccharides.

scholarly journals Proteomics Analysis of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Identified Two New Occlusion-Derived Virus-Associated Proteins, HA44 and HA100

2007 ◽  
Vol 81 (17) ◽  
pp. 9377-9385 ◽  
Author(s):  
Fei Deng ◽  
Ranran Wang ◽  
Minggang Fang ◽  
Yue Jiang ◽  
Xushi Xu ◽  
...  
Keyword(s):  
Helicoverpa Armigera ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Structural Proteins ◽  
Proteomics Analysis ◽  
Group I ◽  
Associated Proteins ◽  
Helicoverpa Armígera ◽  
Group Ii ◽  
First Time

ABSTRACT Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the structural proteins of the occlusion-derived virus (ODV) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV), a group II NPV. Twenty-three structural proteins of HearNPV ODV were identified, 21 of which have been reported previously as structural proteins or ODV-associated proteins in other baculoviruses. These include polyhedrin, P78/83, P49, ODV-E18, ODV-EC27, ODV-E56, P74, LEF-3, HA66 (AC66), DNA polymerase, GP41, VP39, P33, ODV-E25, helicase, P6.9, ODV/BV-C42, VP80, ODV-EC43, ODV-E66, and PIF-1. Two proteins encoded by HearNPV ORF44 (ha44) and ORF100 (ha100) were discovered as ODV-associated proteins for the first time. ha44 encodes a protein of 378 aa with a predicted mass of 42.8 kDa. ha100 encodes a protein of 510 aa with a predicted mass of 58.1 kDa and is a homologue of the gene for poly(ADP-ribose) glycohydrolase (parg). Western blot analysis and immunoelectron microscopy confirmed that HA44 is associated with the nucleocapsid and HA100 is associated with both the nucleocapsid and the envelope of HearNPV ODV. HA44 is conserved in group II NPVs and granuloviruses but does not exist in group I NPVs, while HA100 is conserved only in group II NPVs.

scholarly journals Isolation and Purification of a Cyclooxygenase-2 from the Blood of a Patient Suffering from Rheumatoid Arthritis and Studying the Effect of Natural Products of the Soapwort on the Activity of Purified Enzyme

2016 ◽  
Vol 13 (1) ◽  
pp. 133-145
Author(s):  
Baghdad Science Journal
Keyword(s):  
Rheumatoid Arthritis ◽  
Molecular Weight ◽  
Natural Products ◽  
Negative Impact ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cyclooxygenase 2 ◽  
Isolation And Purification ◽  
Rheumatoid Arthritis Disease ◽  
Approximate Molecular Weight

In this paper to isolate and study the properties of the cyclooxygenase-2 (EC: 1.14.99.1) enzyme in the blood of a patient suffering from rheumatoid arthritis and study the effect of natural products of the Soapwort on the activity of purified enzyme. The study involves taking 30 ml of blood from an adult woman 40 years old, who suffers from rheumatoid arthritis disease for 13 years. Serum is separated and subjected to a series of purification processes including: precipitation by ammonium sulfate, filtration by centrifugation radiator, dialysis in presence of ammonium bicarbonate, separation using the technology of ion exchange, lipholization and then estimating approximate molecular weight of the enzyme using gel filtration technique and sodium dodecyl sulfate (SDS)-page polyacrylamide gel electrophoresis. The study also includes isolating the natural products of Soapwort plant and study the effect of isolated natural products on the activity of the purified enzyme. The result of the study indicates that cycloxygenase-2 has an approximate molecular weight of 71.5 kDa and that the extracted oil of the Soapwort has a negative impact on the activity of the enzyme (r= -0.824; P=0.006), while flavonoids and Saponin have no such impact (r= -0.565; P=0.113; r= -0.634; P=0.067 respectively).

Isolation and Purification of Enterocin EL1, a Bacteriocin Produced by a Strain of Enterococcus faecium†

1995 ◽  
Vol 58 (8) ◽  
pp. 890-898 ◽  
Author(s):  
WANDA J. LYON ◽  
DENNIS G. OLSON ◽  
ELSA A. MURANO
Keyword(s):  
Enterococcus Faecium ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Producer Strain ◽  
Isolation And Purification ◽  
Heat Stable ◽  
Listeria Ivanovii ◽  
Extraction Buffer ◽  
Approximate Molecular Weight

A meat isolate, identified as Enterococcus faecium L1, was found to produce a bacteriocin designated enterocin EL1 Enterocin EL1 was active against a narrow spectrum of microorganisms, inhibiting all tested strains of Listeria. Identification of the producer strain was determined phenotypically by biochemical and morphological tests. Enterocin EL1 was heat stable, sensitive to several proteolytic enzymes, and stable from pH 2 to 11. Adsorption of the bacteriocin to producer cells was dependent on ionic interaction of the bacteriocin and the cell surface at various pHs. By changing the pH of the extraction buffer, enterocin EL1 was extracted from E. faecium L1 cells in a concentrated form. Enterocin EL1 isolated by cell extraction was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a protein with an approximate molecular weight of 2,300. Partially purified enterocin EL1 added to sensitive cells of Listeria ivanovii was bactericidal; however, the bacteriocin did not inhibit the producer strain L1.

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