Purification and characterization of a repressor for the Bacillus cereus glnRA operon.

We report the overexpression, purification, and properties of the regulatory protein, GlnR, for glutamine synthetase synthesis of Bacillus cereus. The protein was found to be a dimer with a molecular weight of approximately 30,000, and its subunit molecular weight was 15,000 in agreement with that (15,025) of deduced amino acid sequence of GlnR. The purified GlnR protein bound specifically to the promoter region of the glnRA operon of B. cereus and Bacillus subtilis. The binding of the GlnR protein to the DNA fragment was enhanced by the presence of glutamine synthetase, the product of glnA, of B. cereus or B. subtilis, although the affinity of the GlnR protein for DNA was not affected in the presence of glutamate, glutamine, Mg2+, Mn2+, or ammonia. These results indicate the existence of an interaction between GlnR and glutamine synthetase, and support the hypothesis that the regulation of glnA expression requires both GlnR protein and glutamine synthetase in Bacillus.     

Purification and characterization of the deoR repressor of Escherichia coli.

The deoR gene, which encodes the deor repressor protein in Escherichia coli, was fused to the strong Ptrc promoter in plasmid pKK233-2. The Ptrc promoter is kept repressed by lacI repressor to prevent cell killing. Induction of the Ptrc--deoR fusion plasmid resulted in the accumulation of 4% of the soluble protein as deoR protein. The deoR repressor protein was purified to 80% purity using conventional techniques; it has a mass of 28.5 kd and appears to exist as an octamer in solution. The deoR repressor is shown by DNase I footprinting to bind to the 16 bp palindromic sequence in the Pribnow box region of the deoP1 promoter. Also, the deoR repressor binds cooperatively in vitro to a DNA template with two deoR binding sites separated by 224 bp in keeping with the conclusion from genetic experiments that more than one operator is required for efficient repression of the deo operon.     

Purification of the Escherichia coli purine regulon repressor and identification of corepressors.

The Escherichia coli pur regulon repressor protein was overproduced in a phage T7 expression system. The overexpressed repressor constituted approximately 35% of the soluble cellular protein. Pur repressor was purified to near homogeneity by two chromatographic steps. Hypoxanthine or guanine was required for binding of purified repressor to purF operator DNA. Apparent dissociation constants of 3.4 nM were determined for binding of holorepressor to purF operator and of 1.7 and 7.1 microM were determined for aporepressor interaction with guanine and hypoxanthine, respectively. A requirement for hypoxanthine or guanine for conversion of aporepressor to holorepressor in vitro supports the earlier report (U. Houlberg and K.F. Jensen, J. Bacteriol. 153:837-845, 1983) that these purine bases are involved in regulation of pur gene expression in Salmonella typhimurium and confirms that hypoxanthine and guanine are corepressors.     

Purification and characterization of repressor of temperate S. aureus phage phi11

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage phi11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A approximately 19 kDa protein copurified with intact His-CI (approximately 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At approximately 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi11 cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators O(L) and O(R), respectively. Equilibrium binding studies indicate that His-CI binds to O(R) with a little more strongly than O(L) and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42 degrees C). Both O(L) and O(R) harbor a nearly identical inverted repeat and studies show that phi11 repressor binds to each repeat efficiently. Additional analyses indicate that phi11 repressor, like lambda repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi11 CI even nearly resembles to that of lambda, phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of phi11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.     

Purification and characterization of the Ner repressor of bacteriophage Mu

The Ner protein of bacteriophage Mu acts as a lambda cro-like negative regulator of the phage's early (transposase) operon. Using the band retardation assay to monitor ner-operator-specific DNA-binding activity, the 8 kDa Ner protein was purified to homogeneity. DNase I footprinting revealed that the purified protein bound and protected a specific DNA operator that contains two 12 bp sites with the consensus sequence 5'-ANPyTAPuCTAAGT-3', separated by a 6 bp spacer region. Moreover, regions corresponding to a turn of the DNA helix flanking these 12 bp repeats are also protected by Ner. Unlike the functionally similar lambda cro protein, gel filtration experiments show that the native molecular mass of Mu Ner to be approx. 8 kDa. These results, plus the pattern of DNase I protection, suggest that the protein may bind as a monomer to each of its specific DNA substrates.     

Purification and characterization of the thyrotropin-releasing hormone (TRH)-degrading serum enzyme and its identification as a product of liver origin.

Previous biochemical studies have indicated that the membrane-bound thyrotropin-releasing hormone (TRH)-degrading enzyme (TRH-DE) from brain and liver and the serum TRH-DE are derived from the same gene. These studies also suggested that the serum enzyme is of liver origin. The present study was undertaken to verify these hypotheses. In different species, a close relationship between the activities of the serum enzyme and the particulate liver enzyme was noticed. The activity of the serum enzyme decreased when rats were treated with thioacetamide, a known hepatotoxin. With hepatocytes cultured in a sandwich configuration, release of the TRH-DE into the culture medium could also be demonstrated. The trypsin-solubilized particulate liver TRH-DE and the serum TRH-DE were purified to electrophoretic homogeneity. Both enzymes and the brain TRH-DE were recognized by a monoclonal antibody generated with the purified brain enzyme as antigen. Lectin blot analysis indicated that the serum enzyme and the liver enzyme are glycoproteins containing a sugar structure of the complex type, whereas the brain enzyme exhibits an oligomannose/hybrid glycostructure. A molecular mass of 97 000 Da could be estimated for all three enzymes after deglycosylation and SDS/PAGE followed by Western blotting. Fragment analysis of the serum TRH-DE revealed that the peptide sequences correspond to the cDNA deduced amino-acid sequences of the membrane-bound brain TRH-DE, whereby two peptides were identified that are encoded by exon 1. These data strongly support the hypothesis that the TRH-DEs are all derived from the same gene, whereby the serum enzyme is generated by proteolytic cleavage of the particulate liver enzyme.     

Identification of Rev-erbalpha as a physiological repressor of apoC-III gene transcription

Elevated serum levels of triglyceride-rich remnant lipoproteins (TRL) are a major risk factor predisposing a subject to atherosclerosis. Apolipoprotein C-III (apoC-III) is a major constituent of TRL that impedes triglyceride hydrolysis and remnant clearance and, as such, may exert pro-atherogenic activities. In the present study, transient cotransfection experiments in rat hepatocytes in primary culture and rabbit kidney RK13 cells demonstrated that overexpression of Rev-erbalpha specifically decreases basal and HNF-4 stimulated human apoC-III promoter activity. A Rev-erbalpha response element was mapped by promoter deletion, mutation analysis, and gel-shift experiments to a AGGTCA half-site located at position -23/-18 (downstream of the TATA box) in the apoC-III promoter. Finally, Rev-erbalpha-deficient mice displayed elevated serum and liver mRNA levels of apoC-III together with increased serum VLDL triglycerides. Taken together, our data identify Rev-erbalpha as a regulator of apoC-III gene expression, providing a novel, physiological role for this nuclear receptor in the regulation of lipid metabolism.     

Purification and properties of a fucoidan-binding protein from human placenta and its identification as immunoglobulin G.

1. A fucoidan-binding protein from human placenta was purified by affinity chromatography on immobilized fucoidan. 2. Characterization of molecular and immunological properties and peptide mapping indicated that the fucoidan-binding protein is an immunoglobulin G. 3. Cleavage with papain and transblot analysis with labelled fucoidan ascertained binding properties of the F(ab) fragments. 4. The specificity for fucoidan was furthermore substantiated by hapten inhibition of haemagglutination as well as by solid-phase assays with biotinylated fucoidan as ligand. The results emphasized the importance of structural features instead of simple ionic interactions. 5. Chemical modification with group-specific reagents to lysine, arginine, tryptophan, tyrosine and histidine resulted in substantial inactivation, their impact being markedly reduced by the presence of fucoidan in the cases of lysine, arginine and tryptophan.     

Mouse macrophage elastase. Purification and characterization as a metalloproteinase.

Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.