We previously expressed VbhTA constructs for lysate AMPylation assays from the pRSFDuet-1 backbone, a plasmid with two multiple cloning sites that each enable the expression of constructs from a PT7 promoter. Our constructs encoded either the VbhT toxin alone (with an N-terminal hexahistidine (His6-)tag) to be expressed from the first multiple cloning site or had additionally the VbhA antitoxin...

In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as glucosamine(+) and N...

Mussel adhesive proteins, including the 20-plus variants of foot protein type 3 (fp-3), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. Here we report the novel production of a recombinant Mytilus galloprovincialis foot protein type 3 variant A (Mgfp-3A) fused with a hexahistidine affinity ligand in Escherichia coli and its appr...

There is a particular need in protein analysis and purification for specific, functional, and generic methods of protein immobilization on solid supports. Here we describe a double-hexahistidine (His6) tag sequence, comprising two hexahistidines separated by an 11-amino acid spacer, which shows at least 1 order of magnitude stronger binding to Ni-NTA-modified surfaces than a conventional single...

Legionella pneumophila, the causative agent of Legionnaire's disease, uses its type IV secretion system to translocate over 300 effector proteins into host cells. These effectors subvert host cell signaling pathways to ensure bacterial proliferation. Despite their importance for pathogenesis, the roles of most of the effectors are yet to be characterized. Key to understanding the function of ef...

Acidobacterium capsulatum is an acid-tolerant, encapsulated, Gram-negative member of the ubiquitous, but poorly understood Acidobacteria phylum. Little is known about the genetics and regulatory mechanisms of A. capsulatum. To begin to address this gap, we identified the gene encoding the A. capsulatum major sigma factor, rpoD, which encodes a 597-amino acid protein with a predicted sequence hi...

The principal bridge connecting the erythrocyte membrane to the spectrin-based skeleton is established by band 3 and ankyrin; mutations leading to reduced bridge formation or increased bridge rupture result in morphological and mechanical abnormalities. Because membrane mechanical properties are determined in part by the protein interactions that stabilize the membrane, we have evaluated the ra...

The important plant hormone salicylic acid (SA; 2-hydroxybenzoic acid) regulates several key plant responses including, most notably, defence against pathogens. A key enzyme for SA biosynthesis is isochorismate synthase (ICS), which converts chorismate into isochorismate, and for which there are two genes in Arabidopsis thaliana One (AtICS1) has been shown to be required for increased SA biosyn...

Many recombinant proteins are produced as a fusion protein tagging hexahistidine, which has been used for studying their biological function both in vitro and in vivo. Unexpectedly, we observed activation of BALB/c mouse splenocytes when treated with hexahistidine-tagged recombinant proteins. This activation was hexahistidine-specific since the anti-pentahistidine antibody completely neutralize...

Fyn-related kinase (Frk) was first identified using human breast cancer cells. It shares 51% identity with c-Src. Like all members of the Src family, Frk is thought to cause several cancers via dysregulations in signal transduction from cell-surface receptors. The excess activity of Frk on beta-cells has a crucial role in type-I diabetes. A silent mutation at Ile229 conferred a bacterial expres...