Isolation, purification, and partial characterization of type V-A hemagglutinin from Escherichia coli GV-12, O1:H-

1982 ◽  
Vol 152 (2) ◽  
pp. 757-761
Author(s):  
V L Sheladia ◽  
J P Chambers ◽  
J Guevara ◽  
D J Evans
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Amino Terminus ◽  
N Terminus ◽  
Type V

A hemagglutinin which specifically agglutinates human type A erythrocytes (mannose resistant) was isolated from the growth medium of cultures of Escherichia coli GV-12, serotype O1:H-, and purified by chromatography on Bio-Gel A-1.5 and DEAE-Sephadex A-25. The purity of the hemagglutinin was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoelectrophoresis. N-terminus analysis indicated that only asparagine resides on the amino terminus. The native hemagglutinin is an aggregate exhibiting a sedimentation coefficient of 9.25, which corresponds to a molecular weight of approximately 200,000. The monomeric molecular weight was found to be approximately 16,300. Amino acid analysis indicated that the hemagglutinin consists of 131 residues, corresponding to a molecular weight of 13,400.

Purification and Characterization of a High-Molecular-Weight Insecticidal Protein Complex Produced by the Entomopathogenic BacteriumPhotorhabdus luminescens

1998 ◽  
Vol 64 (8) ◽  
pp. 3029-3035 ◽  
Author(s):  
David J. Bowen ◽  
Jerald C. Ensign
Keyword(s):  
Molecular Weight ◽  
Insecticidal Activity ◽  
Protein Complex ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Toxin ◽  
The Family ◽  
Toxin Complex

ABSTRACT Photorhabdus luminescens is a gram-negative enteric bacterium that is found in association with entomopathogenic nematodes of the family Heterorhabditidae. The nematodes infect a variety of soil-dwelling insects. Upon entering an insect host, the nematode releases P. luminescens cells from its intestinal tract, and the bacteria quickly establish a lethal septicemia. When grown in peptone broth, in the absence of the nematodes, the bacteria produce a protein toxin complex that is lethal when fed to, or injected into the hemolymph of, Manduca sexta larvae and several other insect species. The toxin purified as a protein complex which has an estimated molecular weight of 1,000,000 and contains no protease, phospholipase, or hemolytic activity and only a trace of lipase activity. The purified toxin possesses insecticidal activity whether injected or given orally. Analyses of the denatured complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed it to be composed of several protein subunits ranging in size from 30 to 200 kDa. The complex was further separated by native gel electrophoresis into three components, two of which retained insecticidal activity. The purified native toxin complex was found to be active in nanogram concentrations against insects representing four orders of the classInsecta.

scholarly journals Purification and characterization of 3-dehydroquinase from Escherichia coli

1986 ◽  
Vol 239 (3) ◽  
pp. 699-704 ◽  
Author(s):  
S Chaudhuri ◽  
J M Lambert ◽  
L A McColl ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Purification And Characterization ◽  
Gel Permeation

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.

Cloning, Expression and Characterization of β-Glucosidase from L. Delbrueckii Subsp. Delbrueckii in Escherichia coli

2011 ◽  
Vol 301-303 ◽  
pp. 347-351
Author(s):  
Xiu Hong Zhao ◽  
Jie Zeng ◽  
Hai Yan Gao ◽  
Chang Biao Li ◽  
Chang Jiang Liu
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gene Encoding ◽  
Prokaryotic Expression Vector ◽  
Strain Bl21

Gene encoding β-glucosidase was amplified through PCR by using the genome DNA extracted from L .delbrueckii subsp. delbrueckii as a template. The gene encoding β-glucosidase was inserted into a prokaryotic expression vector pET-28a(+) and expressed in E.coli strain BL21(DE3). The gene encoding β-glucosidase was of 1380bp. The nucleotide sequence of the gene encoding β-glucosidase from L. delbrueckii subsp. delbrueckii showed as high as 97.9% homology comparing with that from L. delbrueckii subsp. bulgaricus indicating that the gene encoding β-glucosidase is highly conservative. The enzyme activity was about 34U/mg and the molecular weight of β-glucosidase is about 51 kDa analyzed by SDS-polyacrylamide gel electrophoresis.

scholarly journals Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki Characterization of the molecule and determination of the number of polypeptide chains

1979 ◽  
Vol 183 (2) ◽  
pp. 325-330 ◽  
Author(s):  
E Ilan ◽  
E Daniel
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Sedimentation Equilibrium ◽  
Guanidinium Chloride ◽  
Tadpole Shrimp ◽  
Polypeptide Chains

Haemoglobin from the tadpole shrimp, Lepidurus apus lubbocki, was found to have a sedimentation coefficient (s020,w) of 19.3 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 798000 +/- 20000. The amino acid composition showed the lack of cysteine and cystine residues. A haem content of 3.55 +/- 0.03% was determined, corresponding to a minimal mol.wt. of 17400 +/- 200. The pH-independence in the range pH 5-11 of the sedimentation coefficient indicates a relatively high stability of the native molecule. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a mol.wt. of 34000 +/- 1500. The molecular weight of the polypeptide chain was determined to be 32800 +/- 800 by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol. The findings indicate that Lepidurus haemoglobin is composed of 24 identical polypeptide chains, carrying two haem groups each.

scholarly journals Biochemical Characterization of SFC-1, a Class A Carbapenem-Hydrolyzing β-Lactamase

2007 ◽  
Vol 51 (12) ◽  
pp. 4512-4514 ◽  
Author(s):  
Fátima Fonseca ◽  
Ana Cristina Sarmento ◽  
Isabel Henriques ◽  
Bart Samyn ◽  
Jozef van Beeumen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulfate ◽  
Molecular Mass ◽  
Clavulanic Acid ◽  
Gel Electrophoresis ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Class A

ABSTRACT The carbapenem-hydrolyzing β-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins, cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and tazobactam.

scholarly journals The composition and the structure of bacterioferritin of Escherichia coli

1981 ◽  
Vol 197 (1) ◽  
pp. 171-175 ◽  
Author(s):  
J Yariv ◽  
A J Kalb ◽  
R Sperling ◽  
E R Bauminger ◽  
S G Cohen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Electron Microscope ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Iron Compound ◽  
Protein Subunit ◽  
Electron Dense Core

Bacterioferritin isolated from Escherichia coli is of two kinds: a protein containing a polynuclear iron compound, the bacterioferritin proper and a protein free of the polynuclear iron compound, the apo-bacterioferritin. Bacterioferritin of both kinds is characterized by absorption maxima at 417,530 and 560 nm, contributed by protohaem IX. Single crystals of bacterioferritin of the space group I432 suggest that the molecule is made up of 24 identical subunits related by a cubic point symmetry. The molecular weight of the protein subunit, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, is 15000. In the electron microscope the bacterioferritin molecule appears to be a sphere of 9.5 nm (95 A) diameter composed of a negatively staining outer shell and an inner electron-dense core of 6 nm (60 A) diameter.

Purification and Characterization of an Unusual Aminopeptidase From Pea Seeds

1980 ◽  
Vol 7 (2) ◽  
pp. 131 ◽  
Author(s):  
JB Caldwell ◽  
LG Sparrow
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sulfhydryl Group ◽  
Purification And Characterization ◽  
Apparent Homogeneity ◽  
Pea Seeds ◽  
Hydrolysis Of

An aminopeptidase with specificity for N-terminal glutamic and aspartic acid residues has been purified to apparent homogeneity from pea seeds (Pisum sativum cv. Greenfeast). It also catalyses the hydrolysis of the glutaryl-phenylalanine bond of the synthetic chymotrypsin substrate glutaryl- L-phenylalanine p-nitroanilide. The native enzyme, which has a molecular weight of approximately 500 000, gives a single band on polyacrylamide gel electrophoresis but two major bands when subjected to electrophoresis in the presence of sodium dodecyl sulfate after reduction. Its behaviour with various inhibitors suggests that a sulfhydryl group is important for its activity.

A glyco-phosphoprotein in human milk

1987 ◽  
Vol 54 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Norihiro Azuma ◽  
Kunio Yamauchi
Keyword(s):  
Molecular Weight ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Gel Electrophoresis ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Reversed Phase ◽  
Ultracentrifugal Analysis

SummaryA highly glycosylated phosphoprotein (HGPP) was isolated from a human casein fraction by reversed-phase high-performance liquid chromatography. This component contained carbohydrates to ∼ 38·2% (w/w) and phosphorus to ∼ 1·6% (w/w). The molecular weight of this HGPP as estimated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis ∼ 41000. Ultracentrifugal analysis revealed that the sedimentation coefficient of the HGPP was 2·6S in a 10 mM-imidazole-HCl buffer at pH 7·0 and 27 °C, but this component interacted with human κ-casein and formed a complex with s = 10·4S.

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

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
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).

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

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