The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide.

The affinity system based on the artificial peptide ligand Strep-tag® II and engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide - instead of D-biotin recognized by the natural protein - to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag®. Four representative streptavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology.

Development of the Twin-Strep-tag® and its application for purification of recombinant proteins from cell culture supernatants.

Short peptide affinity tags have become indispensable in protein research. They cannot only be used for affinity purification but also downstream for detection and assay of an arbitrary fused recombinant protein without the need for any prior knowledge of its biochemical properties. Strep-tag®II is particularly popular for providing recombinant proteins at high purity and functionality by using physiological conditions within a rapid one-step protocol. The affinity receptor for Strep-tag®II is affinity engineered streptavidin, named Strep-Tactin®. Strep-tag®II binds to the biotin binding pocket enabling mild competitive elution with biotin derivatives, preferably desthiobiotin, for repeated use of the Strep-Tactin® affinity resins. Fast binding and dissociation kinetics allow comparatively high flow rates throughout column chromatography including elution. Fast dissociation kinetics may be, however, limiting for using Strep-tag®II for direct purification of target proteins from large volumes of diluted extracts like mammalian cell culture supernatants or in assay formats requiring extended washing like ELISA. For this reason, binding characteristics were improved by development of the Twin-Strep-tag® consisting of two Strep-tag®II moieties connected by a short linker. The resulting avidity effect, i.e., the combined synergistic binding of two Strep-tag®II moieties to tetrameric Strep-Tactin®, reduces the off-rate for more steady binding under non-competitive conditions. The addition of a competitor, however, reverses the synergistic avidity effect and, hence, efficient elution capability is preserved. In fact, the Twin-Strep-tag® features all beneficial properties of Strep-tag®II, including efficient elution under gentle competitive conditions, but, due to its higher affinity, additionally enables a more universal use in applications requiring stable binding.

Invasiveness is a variable and heterogeneous phenotype in Listeria monocytogenes serotype strains.

The ability of Listeria monocytogenes to breach mucosal and endothelial barriers of the host during infection is a hallmark property mediated by the internalins (Inl) A and B. We examined the invasive property of several L. monocytogenes strains representing 13 serotypes. We found that invasiveness is a heterogeneous phenotype amongst L. monocytogenes serotype strains. Despite this, many of the poorly invasive and non-invasive strains of L. monocytogenes express internalins at levels comparable to those of invasive isolates. Introduction of the inlAB locus from EGD-e into several poorly invasive strains had no effect on their invasive properties. A strain from serotype 4b that exhibits highly invasive properties was further examined. Deletion of the inlAB locus abrogated invasion of this strain while reintroduction of the inlAB locus into this strain restored invasiveness. An analysis of regions flanking the inlAB locus revealed considerable differences in the strains studied. Our results suggest that efficacious entry of L. monocytogenes into eukaryotic cells is complex and requires additional factors apart from internalins. Data presented here also suggest that the inlAB locus was introduced into L. monocytogenes by horizontal gene transfer with subsequent deletion and rearrangements occurring during evolution of this species.