scholarly journals Interactions of an Essential Bacillus subtilis GTPase, YsxC, with Ribosomes

2007 ◽  
Vol 190 (2) ◽  
pp. 681-690 ◽  
Author(s):  
Catherine Wicker-Planquart ◽  
Anne-Emmanuelle Foucher ◽  
Mathilde Louwagie ◽  
Robert A. Britton ◽  
Jean-Michel Jault
Keyword(s):  
Bacillus Subtilis ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Small Gtpase ◽  
E Coli ◽  
Protein Partners ◽  
The One

ABSTRACT YsxC is a small GTPase of Bacillus subtilis with essential but still unknown function, although recent works have suggested that it might be involved in ribosome biogenesis. Here, purified YsxC overexpressed in Escherichia coli was found to be partly associated with high-molecular-weight material, most likely rRNA, and thus eluted from gel filtration as a large complex. In addition, purification of ribosomes from an E. coli strain overexpressing YsxC allowed the copurification of the YsxC protein. Purified YsxC was shown to bind preferentially to the 50S subunit of B. subtilis ribosomes; this interaction was modulated by nucleotides and was stronger in the presence of a nonhydrolyzable GTP analogue than with GTP. Far-Western blotting analysis performed with His6-YsxC and ribosomal proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that YsxC interacted with at least four ribosomal proteins from the 50S subunit. Two of these putative protein partners were identified by mass spectrometry as L1 and L3, while the third reactive band in the one-dimensional gel contained L6 and L10. The fourth band that reacted with YsxC contained a mixture of three proteins, L7/L12, L23, and L27, suggesting that at least one of them binds to YsxC. Coimmobilization assays confirmed that L1, L6, and L7/L12 interact with YsxC. Together, these results suggest that YsxC plays a role in ribosome assembly.

II. Mitt.: Auflösung der Chromoproteidfraktion aus Rattenleber-Ribosomen mit Hilfe der Polyacrylamidgel-Elektrophorese

1970 ◽  
Vol 25 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Wolfram Domschke ◽  
Jürgen G. Meyer-Bertenrath
Keyword(s):  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
The Other ◽  
Blue Staining ◽  
Ribosomal Subunits ◽  
Molecular Weights ◽  
The One ◽  
Liver Ribosomes

After preparation of a coloured protein component containing iron from rat liver ribosomes 1 this fraction was submitted to detailed analysis by means of polyacrylamide gel electrophoresis. Thus it may be separated into one main and two secondary bands, which do not contain RNA detectable by methylene blue staining. The ferric content of all bands can be demonstrated by staining with 2,4-dinitroso-1,3-naphthalenediol 2. These bands are to be found in the large ribosomal subunits as well as in the small one in qualitative conformity, they differ, however, in their quantitative relations to each other depending on origin. LiCl-extraction as described for the preparation of ribosomal proteins causes dissociation of the chromoproteid fraction into six bands possessing lower molecular weights each than the original bands.The chromoproteids, localized in the large ribosomal subunits on the one hand and in the small subunits on the other hand were prepared differentiatedly by gel filtration. Results show the chromoproteid represented in the 57-S-subunit on a bigger scale than the nucleoproteid part, on the contrary, the 29-S-subunit is constructed of RNA-containing material preferably.

scholarly journals Expression and Purification of the Recombinant Lethal Factor of Bacillus anthracis

1998 ◽  
Vol 66 (2) ◽  
pp. 862-865 ◽  
Author(s):  
Pankaj Gupta ◽  
Smriti Batra ◽  
Arun P. Chopra ◽  
Yogendra Singh ◽  
Rakesh Bhatnagar
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Lethal Factor ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Nitrilotriacetic Acid ◽  
Biologically Active ◽  
Liquid Chromatograph ◽  
E Coli

ABSTRACT The structural gene for the 90-kDa lethal factor (LF) isolated fromBacillus anthracis was expressed as a fusion protein with six histidine residues in Escherichia coli. Expression of LF in E. coli under the transcriptional regulation of the T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 90 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant LF reacted with anti-LF antibodies. The protein was purified to homogeneity by nickel nitrilotriacetic acid affinity chromatography and gel filtration on a Sephacryl S-200 column followed by anion exchange on a fast-performance liquid chromatograph with a Resource-Q column. The yield of purified LF from this procedure was 1.5 mg/liter. In solution, trypsin cleaved protective antigen bound to native and recombinant LF with comparable affinities. In macrophage lysis assays, native and recombinant LF exhibited identical potencies. The results suggest that large amounts of biologically active LF can be purified by this procedure.

scholarly journals The Effect of Single Co-expression of The DnaK-DnaJ-GrpE and GroEL/ES Chaperones and Their Combination on Expression Intein-pretrombin-2 in Escherichia coli ER2566

2020 ◽  
Vol 6 (1) ◽  
pp. 47-54
Author(s):  
Iman Permana Maksum ◽  
D Agus Yusuf Wildan ◽  
Khomaini Hasan ◽  
Toto Subroto
Keyword(s):  
Inclusion Bodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Iptg Induction ◽  
E Coli ◽  
Expression Host ◽  
Sds Page ◽  
Culture Growth ◽  
The One ◽  
Cell Culture Growth

The use of recombinant thrombin in the manufacture of fibrin glue allows diseases contamination to be avoided. However, the expression of recombinant protein in E. coli still has a disadvantage of the formation of inclusion bodies, so it needs to be minimized by co-expression of chaperones. Therefore, the aim of this study was to determine the effect of single DnaK-DnaJ-GrpE and GroEL/ES chaperone expression and their combination on the expression of intein-pretrombin-2Ti,pH on E. coli ER2566. The method started with isolation of pTWIN1-prethrombin-2Ti,pH and pG-KJE8 from E. coli TOP10F' and DH5α respectively, the co-transformation of the expression host E. coli ER2566 using pG-KJE8 and pTWIN1-prethrombin-2Ti,pHvectors, the chaperone co-expression was induced using L-Arabinosa before IPTG induction and cell culture growth was incubated at 22 oC. The expression products were characterized by using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results of the co-expression of chaperone showed that the number of soluble fraction was higher than the one without co-expression of chaperone. In addition, the co-expression of chaperone using pG-KJE8 in intein-prothrombin-2Ti,pH expression was sufficient using tetracycline as an inducer.

scholarly journals Purification and characterization of methyl-accepting chemotaxis protein methyltransferase I in Bacillus subtilis

1981 ◽  
Vol 199 (3) ◽  
pp. 795-805 ◽  
Author(s):  
A H J Ullah ◽  
G W Ordal
Keyword(s):  
Bacillus Subtilis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Single Species ◽  
Competitive Inhibitor ◽  
Sensory Stimuli ◽  
Molecular Weights ◽  
Protein Methyltransferase

A methyltransferase that methylates one of the proteins involved in chemotactic adaptation to sensory stimuli in Bacillus subtilis was purified to homogeneity. The enzyme utilizes S-adenosylmethionine as donor for a methyl group that is transferred to a glutamate residue in a 69 000-mol.wt. membrane protein and also to a protein of 19 000 mol.wt. The molecular weights of the denatured enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of the native enzyme by gel-filtration chromatography both show the protein to be a 44 000-mol.wt. monomer. Isoelectric focusing of the purified methyltransferase showed the protein to be a single species with isoelectric point pI 5.4. On the basis of a molecular weight of 44 000, the molar absorption coefficient at 262 nm of the enzyme is 10.9 x 10(4) M-1 . cm-1. The Km of the enzyme for S-adenosylmethionine is about 2 microM. The Ki for S-adenosylhomocysteine is about 0.2 microM. Ca2+ is a competitive inhibitor of methylation, with a Ki of 0.065 microM. The enzyme methylates membranes from the wild-type more efficiently than membranes isolated from a mutant strain defective in chemotaxis. The enzyme is unable to methylate Escherichia coli membranes.

scholarly journals Restoration of β-galactosidase to Escherichia coli M15. Complementation studies

1977 ◽  
Vol 165 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Dobrivoje V. Marinkovic ◽  
Jelka N. Marinkovic
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Galactosidase Activity ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Molecular Weights ◽  
Terminal Amino

Carboxymethylated β-galactosidase from Escherichia coli was dissociated at 100°C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore β-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ‘complementable fractions’. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the β-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented β-galactosidase can exist.

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.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
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 (SDS–PAGE). The incorporation of gelatin into SDS–PAGE 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.

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.

Sign in / Sign up

Export Citation Format

Share Document