Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, S...

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the ...

Natural proteins are often confined within their local microenvironments, such as three-dimensional confinement in organelles or two-dimensional confinement in lipid rafts on cytoplasmic membrane. Spatial confinement restricts proteins' entropic freedom, forces their lateral interaction, and induces new properties that the same proteins lack at the soluble state. So far, the phenomenon of envir...

The αvβ6 integrin is up-regulated in cancer and wound healing but it is not generally expressed in healthy adult tissue. There is increasing evidence that it has a role in cancer progression and will be a useful target for antibody-directed cancer therapies. We report a novel recombinant diabody antibody fragment that targets specifically αvβ6 and blocks its function. The diabody was engineered...

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...