Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent cond...

Knowledge of the response of cytochrome P450 1B1 (CYP1B1) to exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in both humans and rodents is limited. To improve the analysis of CYP1 proteins, specific CYP1B1 and CYP1A1 polypeptides were expressed as hexahistidine-tagged fusion proteins in Escherichia coli, purified to homogeneity and used to produce polyclonal antibodies in rabbits. Immuno...

Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as t...

Copper coordination to soluble oligomers of the English (AβH(6)R) mutant of the amyloid-β peptide is probed. Cu(II) coordination yields a square planar (N/O)4 coordination environment, while reduction yields an O2 inert linear bis-His Cu(I) centre.

Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae. The open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that was postulated to be involved in the replication and the formation of cap structure at the 5' end of the viral genome. To characterize the activities associated with the 155-kDa viral protein, it was expressed in ...

In vitro selection targeting an anti-polyhistidine monoclonal antibody was performed using mRNA display with a random, unconstrained 27-mer peptide library. After six rounds of selection, epitope-like peptides were identified that contain two to five consecutive, internal histidines and are biased for arginine residues, without any other identifiable consensus. The epitope was further refined b...

A hexahistidine-tagged C-terminal nucleotide-binding domain (H6-NBD2) from mouse P-glycoprotein was designed, overexpressed, and purified as a highly soluble recombinant protein. Intrinsic fluorescence of its single tryptophan residue allowed monitoring of high-affinity binding of 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP), a fluorescent ATP derivative that induces a marked quenching correlated...

Recombinant subunit vaccines consisting of the F1 and V antigens of Yersinia pestis have shown remarkable promise for protection of animals and humans against bubonic and pneumonic plague. Previously we demonstrated that intranasal administration of a recombinant protein consisting of the ricin B chain (RTB) fused with an antigen purified from transgenic Nicotiana tabacum (tobacco) hairy root c...

Streptococcus sobrinus, one agent of dental caries, secretes a protein that induces lymphocyte polyclonal activation of the host as a mechanism of immune evasion. We have isolated from culture supernatants of this bacterium a protein with murine B-cell-stimulatory properties and subsequently cloned the relevant gene. It contains an open reading frame of 825 bp encoding a polypeptide with 275 am...