[40] High-sensitivity sequence analysis of proteins recovered from sodium dodecyl sulfate gels

As, Bhown; Jc, Bennett

doi:10.1016/s0076-6879(83)91042-x

Cited by 37 publications

(16 citation statements)

References 7 publications

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“…Since the purified preparations stagnated in the stacking gel during SDS-PAGE, the purity and activity of the purified enzymes were determined by non-denaturing PAGE, which was performed according to the method of Bhown & Bennett (1983). All buffers and solutions were made fresh, stored at 4 8C and used within a week to obtain the most consistent results.…”

Section: Methodsmentioning

confidence: 99%

Cloning of the Tannerella forsythensis (Bacteroides forsythus) siaHI gene and purification of the sialidase enzyme

Ishikura

Arakawa

Nakajima

et al. 2003

Journal of Medical Microbiology

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Tannerella forsythensis (previously named Bacteroides forsythus) is a Gram-negative, anaerobic, fusiform bacterium that is a primary or secondary aetiological agent in periodontal disease in humans. T. forsythensis expresses several putative virulence factors, including a sialidase; however, there has been no molecular genetic characterization of this enzyme. A sialidase clone (pHI-1) was screened from a total of 455 recombinant clones of a genomic DNA library using the 29-(4-methylumbelliferyl)-AE-D-N-acetylneuraminic acid (MUNeuAc) filter-paper spot assay. The sialidase gene ORF (siaHI) consists of a 1395 bp coding sequence and encodes a protein with 465 amino acids with an overall molecular mass of 52 kDa. The sialidase does not have sequence similarity to any other bacterial sialidase. The entire sialidase ORF was expressed in Escherichia coli. Furthermore, the sialidase was purified from the type strain of T. forsythensis and from a recombinant clone, pHI-1 : 1, and was analysed using a non-denaturing gel, revealing that the enzyme preparations were respectively separated as two major bands and as a single band. Southern blot hybridization analysis revealed similar patterns of siaHI-hybridizing bands among clinical isolates of T. forsythensis from periodontitis patients. This is the first study on the cloning and expression of a T. forsythensis sialidase gene and the purification of the SiaHI enzyme from T. forsythensis ATCC 43037 T and recombinant E. coli.

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Section: Methodsmentioning

confidence: 99%

Cloning of the Tannerella forsythensis (Bacteroides forsythus) siaHI gene and purification of the sialidase enzyme

Ishikura

Arakawa

Nakajima

et al. 2003

Journal of Medical Microbiology

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“…To purify the fusion protein, tryptophan-starved bacterial cells carrying the plasmid were induced for trpE expression (58), the insoluble protein fraction was prepared essentially as described (26), and proteins were subjected to preparative electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide (7.5%). The fusion protein was visualized with cold 0.25 M KCI, excised from the gel, and electroeluted (2).…”

Section: Methodsmentioning

confidence: 99%

The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae.

Schultz¹,

Marshall-Carlson²,

Carlson³

1990

Mol. Cell. Biol.

112

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The SSN6 protein functions as a negative regulator of a variety of genes in Saccharomyces cerevisiae and is required for normal growth, mating, and sporulation. It is a member of a family defined by a repeated amino acid sequence, the TPR (tetratricopeptide repeat) motif. Here, we have used specific antibody to identify and characterize the SSN6 protein. Both SSN6 and a bifunctional SSN6-j0-galactosidase fusion protein were localized in the nucleus by immunofluorescence staining. The N-terminal one-third of the protein containing the TPR units was identified as the region that is important for SSN6 function. Analysis of four nonsense alleles, isolated as intragenic suppressors of an ssn6::URA3 insertion, revealed that polypeptides truncated after TPR unit 7 provide SSN6 function. Deletion analysis suggested that TPR units are required but that 4 of the 10 TPR units are sufficient. In addition, deletion studies indicated that three very long, homogeneous tracts of polyglutamine and poly(glutamine-alanine) are dispensable. Previous genetic evidence suggested the SSN6 protein as a possible target of the SNF1 protein kinase. Here, we show that the C terminus of SSN6 is phosphorylated in vivo and that the SNF1 kinase is not responsible for most of the phosphorylation. Finally, SSN6 has a modest effect on the maintenance of minichromosomes.The Saccharomyces cerevisiae SSN6 gene encodes a protein that functions as a negative regulator of gene expression with a broad range of action and that is required for normal growth. Mutations at the locus cause diverse pleiotropic phenotypes, suggesting that SSN6 affects the expression of many genes (6,52,63). The ssn6 mutants show slow growth at 30°C, temperature sensitivity for growth, extreme clumpiness, defects in utilization of glycerol, and high-level, glucose-insensitive expression of SUC2 and other glucoserepressible genes. MATa ssn6 strains exhibit mating defects because of failure to repress genes that are normally expressed only in MATa strains. Homozygous mutant diploids are defective in sporulation. In addition, ssn6 is allelic to cyc8, which causes overproduction of iso-2-cytochrome c (49). SSN6 affects SUC2 (invertase) gene expression at the transcriptional level, and overexpression of the SSN6 gene prevents full derepression of SUC2, which is consistent with a role for the SSN6 protein as a negative regulator of SUC2 (52). Taken together, this evidence indicates that SSN6 has a role in regulating expression of genes with a variety of functions and that SSN6 is important for normal cell growth, mating, and sporulation.Sequence analysis of the SSN6 gene predicted a 107-kilodalton (kDa) product with several unusual structural features (52, 64). Near its N terminus, the SSN6 protein includes 10 tandem repeats of a 34-amino-acid sequence, termed the TPR (tetratricopeptide repeat) motif, which was recently identified by Sikorski et al. (54). This repeated sequence defines a family of six genes, from several organisms, that encode structurally similar proteins. In additio...

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“…The acrylamide concentration in the 1-4 mm-thick and 140 mm-long separating gel was 5 to 15 ~. Electrophoresis was at 60 V for 18 h. After electrophoresis the proteins were localized by dipping the gel into 0.1 M-KC1 at 4 °C for about 5 min (Bhown & Bennett, 1983). Protein-containing visible gel slices were cut out, finely divided and eluted with 4 ml (about 5 times the volume of the wet gel slice) 0.1 ~ SDS by slow end-over-end agitation at 30 °C for 18 h. The filtered eluates of E1 and capsid proteins were made 16~o in TCA, kept for 3 h at 0 °C and centrifuged at 10000g for 20 min at 4 °C.…”

Section: Introductionmentioning

confidence: 99%

Three Genes Code for Rubella Virus Structural Proteins E1, E2a, E2b and C

Kalkkinen

Oker‐Blom

Pettersson

1984

Journal of General Virology

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SUMMARYThe structural proteins E 1, E2a, E2b and C of rubella virus (RV) were purified by preparative SDS-PAGE. The individual proteins were subjected to amino-terminal sequence analysis by Edman degradation, carboxyl-terminal structure analysis by digestion with carboxypeptidases and quantitative amino acid composition analysis. The partial amino-terminal sequences of E2a and E2b were identical and different from that of El. The C protein did not yield any consistent results on Edman degradation, suggesting that its amino-terminus is blocked. The amino acid compositions of E2a and E2b were very similar and differed from that obtained for E1 and C, which also differed from each other. Carboxypeptidase digestions showed that E2a and E2b have an identical carboxyl-terminal structure, which differed from that of the C protein. No amino acid residues were released from the E1 protein by digestion with a mixture of carboxypeptidases A and B. These results confirm that the structural proteins of RV are translated from three genes corresponding to C, E2 and El. E2 exists in virions in two post-translationally modified forms, E2a and E2b, which have an identical apoprotein moiety. The partial amino acid sequence information obtained here should also be sufficient to localize the ends of the individual genes on the 24S mRNA genuine once its nucleotide sequence has been established.

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[40] High-sensitivity sequence analysis of proteins recovered from sodium dodecyl sulfate gels

Cited by 37 publications

References 7 publications

Cloning of the Tannerella forsythensis (Bacteroides forsythus) siaHI gene and purification of the sialidase enzyme

Cloning of the Tannerella forsythensis (Bacteroides forsythus) siaHI gene and purification of the sialidase enzyme

The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae.

Three Genes Code for Rubella Virus Structural Proteins E1, E2a, E2b and C

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