scholarly journals Inosine 5′-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5′-monophosphate

1979 ◽  
Vol 183 (3) ◽  
pp. 481-494 ◽  
Author(s):  
H J Gilbert ◽  
C R Lowe ◽  
W T Drabble
Keyword(s):  
Escherichia Coli ◽  
Affinity Chromatography ◽  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Competitive Inhibitor ◽  
Subunit Structure ◽  
Imp Dehydrogenase ◽  
Finger Printing ◽  
Monospecific Antisera

Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic ‘finger-printing’ demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli.

scholarly journals Media Optimization for Production of Recombinant Carrier Protein (CRM197) in Escherichia coli

2021 ◽  
Vol 13 (1) ◽  
pp. 283-297
Author(s):  
S. Shukla ◽  
D. Mishra
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Luria Bertani ◽  
Lag Phase ◽  
Carrier Protein ◽  
Scale Production ◽  
E Coli ◽  
Defined Media

Since the advent of vaccines, the mankind has benefited from the same and has been able to curb the mortality rate around the globe. Amongst different types of available vaccines, polysaccharide based vaccines are very widely used against various infectious diseases. The polysaccharide vaccines need to be conjugated with a carrier protein to make the vaccine more immunogenic. Recombinant Escherichia coli cells are the organism of choice for large scale production of a carrier protein because of its widely studied scientific aspects. In the present study, for proof of concept, the recombinant E. coli cells were cultured in Luria-Bertani media to check the expression of rCRM197. At 80L scale, it was observed that when recombinant E. coli cells were grown in a chemically defined media, it resulted in inconsistent growth and a long lag phase. When the defined media was supplemented with yeast extract, the lag phase of the culture was substantially reduced and the maximum growth of the culture was achieved. Protein expression was checked using SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) and Western blot technique. The optimized media resulted in a robust fermentation process to achieve high cell density and maximum biomass for the production of recombinant protein.

Affinity Chromatography of Factor VIII Subunits

1979 ◽  
Author(s):  
J. L. Lane ◽  
H. Ekert
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Functional Properties ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Antibody ◽  
Subunit Structure ◽  
Similar Treatment ◽  
Human Factor Viii ◽  
Sds Page

Gel filtration of factor VIII resulted in elution of a fraction which possessed procoagulant activity (VIII:C), ristocetin cofactor (VIIIrRCF) and antigenic activity (VIII:Rag). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of this preparation showed that no protein entered the gel. Eight bands ranging from 30,000 to 230,000 molecular weight (mw) were seen when 400μg reduced protein was loaded onto the SDS-PA geL Attempts to assay the functional properties were made in order to investigate whether the multiband structure was a property of factor VIII or due to contaminants. These proved unsuccessful, as reduction and SDS-PAGE of factor VIII destroyed its functional properties. Affinity chromatography with human and rabbit antibodies to factor VIII provided an alternative method of relating the subunit structure to functional properties. Insolubilized human antibody inactivated VIII:C and did not bind VIII:Rag. The protein bound to human antibody was dissociated with NH SCN and reduced. SDS-PAGE showed heavy protein staining, with 95% of the protein present in 35,000 and 62,000 mw bands. Similar treatment of a control human IgG column showed minimal protein in the same mw region. Using insolubilized rabbit antibody to human factor VIII, all 8 bands were observed after N^SCN dissociation, reduction and SDS-PAGE. These results suggest that contaminat^ely that the low bands observed on heavily loaded gels are due to protein contamination.

Affinity Chromatography of Factor VIII Subunits

1979 ◽  
Author(s):  
J.L. Lane ◽  
H. Ekert
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Functional Properties ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Antibody ◽  
Subunit Structure ◽  
Similar Treatment ◽  
Human Factor Viii ◽  
Sds Page

Gel filtration of factor VIII resulted in elution of a fraction which possessed procdegulant activity (VIII:C), ristocetin cofactor (VIII:RCF) and antigenic activity (VIII:Rag). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of this preparation showed that no protein entered the gel. Eight bands ranging from 30,000 to 230,000 molecular weight (mw) were seen when 400μg reduced protein was loaded onto the SDS-PA geL Attempts to assay the functional properties were made in order to investigate whether the multiband structure was a property of factor VIII or due to contaminants. These proved unsuccessful, as reduction and SDS-PAGE of factor VIII destroyed its functional properties. Affinity chromatography with human and rabbit antibodies to factor VIII provided an alternative method of relating the subunit structure to functional properties. In-solubilized human antibody inactivated VIII:C and did not bind VIII. Rag. The protein bound to human antibody was dissociated with NH4 SCN and reduced. SDS-PAGE showed heavy protein staining, with 95% of the protein present in 35,000 and 62,000 mw bands. Similar treatment of a control human IgG column showed minimal protein in the same mw region. Using insolubilized rabbit antibody to human factor VIII, all 8 bands were observed after NH4 SCN dissociation, reduction and SDS-PAGE. These results suggest that it is unlikely that the low mw bands observed on heavily loaded gels are due to protein contamination.

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

1986 ◽  
Vol 64 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Malcolm B. Perry ◽  
Leann MacLean ◽  
Douglas W. Griffith
Keyword(s):  
Escherichia Coli ◽  
Brucella Abortus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Periodate Oxidation ◽  
Cross Reactivity ◽  
E Coli ◽  
Structure Formula ◽  
Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

Affinity chromatography purification and partial characterization of phytohemagglutinin-receptor glycoproteins from porcine splenic lymphocytes

1985 ◽  
Vol 63 (9) ◽  
pp. 932-940 ◽  
Author(s):  
Gilles Dupuis ◽  
Jean-Pierre Doucet ◽  
Bânû Bastin ◽  
Jeannine Cardin
Keyword(s):  
Affinity Chromatography ◽  
Competitive Inhibition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Interleukin 2 ◽  
Lymphocyte Activation ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Murine Cell Line ◽  
Lymphocyte Cultures

We describe the isolation of pig spleen lymphocyte glycoproteins that interact with phytohemagglutinin (PHA), the lectin from Phaseolus vulgaris. Purification was achieved by affinity chromatography of a Nonidet P-40 extract of the cells on a PHA – Affi-Gel 10 column. The retained glycoproteins were eluted with an acidic (pH 3.0) glycine buffer and represented 1.9–2.4% of the amount of protein applied to the column. They contained 20 ± 1.3% hexose and 1.7 ± 0.7% fatty acids, on a weight basis. Electrophoretic analyses (sodium dodecyl sulfate – polyacrylamide gel electrophoresis) showed the presence of major Coomassie blue positive bands with apparent molecular masses of 50–55, 75, 95, 130, and 155 kdaltons along with minor bands of 20–40, 42, 45, 60–65, 175, and 200–250 kdaltons. The purified PHA-receptor glycoproteins inhibited, in a dose-dependent manner, the incorporation of [3H]thymidine in pig lymphocytes cultured at a concentration of 106 cells/mL in the presence of PHA. A 50% inhibition was observed when 20 μg/mL of the glycoproteins was added to the lymphocyte cultures containing 0.5 μg/mL of PHA. Scatchard analysis of the binding of 125I-labelled PHA, in the presence of increasing amounts of the purified glycoproteins, showed a suppression of the binding of the lectin to high affinity sites of the cells, as evidenced by a change from biphasic to a linear profile. Results of binding suggested a competitive inhibition by a population of purified glycoproteins with a similar affinity for the lectin. The purified glycoproteins decreased PHA-dependent interleukin 2 (IL-2) production by pig lymphocytes as assayed with a IL-2 dependent murine cell line. It is suggested that the affinity-purified PHA-reactive glycoproteins are inhibitors of PHA-dependent cellular responses because they compete with PHA-receptor sites on the lymphocyte plasma membrane. A mouse antiserum raised against the purified glycoproteins inhibited PHA-induced lymphocyte activation, but did not stimulate lymphocytes when added alone to lymphocyte cultures or in combination with a antimouse antiserum.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Pseudomonas putida

1986 ◽  
Vol 64 (12) ◽  
pp. 1288-1293 ◽  
Author(s):  
Josefa M. Alonso ◽  
Amando Garrido-Pertierra
Keyword(s):  
Pseudomonas Putida ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Catabolic Pathway ◽  
Ph Optimum ◽  
Semialdehyde Dehydrogenase ◽  
Standard Assay ◽  
Assay Conditions

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMSA) dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway was purified from Pseudomonas putida by gel filtration, anion-exchange, and affinity chromatographies. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis suggested an approximate tetrameric molecular weight of 200 000. The purified enzyme showed a pH optimum at 7.8. The temperature–activity relationship for the enzyme from 27 to 45 °C showed broken Arrhenius plots with an inflexion at 36–37 °C. Under standard assay conditions, the enzyme acted preferentially with NAD. It could also catalyze the reduction with NADP (which had a higher Km), at 18% of the rate observed for NAD. The following kinetic parameters were found: Km(NAD) = 20.0 ± 3.6 μM, Km(CHMSA) = 8.5 ± 1.8 μM, and Kd(enzyme–NAD complex) = 7.8 ± 2.0 μM. The product NADH acted as a competitive inhibitor against NAD.

scholarly journals Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degPMutants and Induces the Cpx and ςE Extracytoplasmic Stress Responses

2001 ◽  
Vol 183 (18) ◽  
pp. 5230-5238 ◽  
Author(s):  
Geoffrey R. Langen ◽  
Jill R. Harper ◽  
Thomas J. Silhavy ◽  
S. Peter Howard
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Stress Responses ◽  
Elevated Temperatures ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phospholipase A ◽  
Temperature Sensitive ◽  
Outer Membrane Phospholipase A ◽  
Temperature Sensitive Phenotype

ABSTRACT DegP is a periplasmic protease that is a member of both the ςE and Cpx extracytoplasmic stress regulons ofEscherichia coli and is essential for viability at temperatures above 42°C. [U-14C]acetate labeling experiments demonstrated that phospholipids were degraded indegP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, β-lactamase, and β-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degPcells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and ςE regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype ofdegP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and ςE regulons rather than by inactivating the phospholipase per se.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

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