The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG (R)- or myc-tag. The ALFA-tag forms a small and stable a-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFA(PE)) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions

Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer

Epitope tags are widely employed as tools to detect, purify and manipulate proteins in various experimental systems. We recently introduced the ALFA-tag together with two ALFA-specific single-domain antibodies (sdAbs), NbALFA and NbALFAPE, featuring high or intermediate affinity, respectively. Together, the ALFA system can be employed for a broad range of applications in microscopy, cell biology and biochemistry requiring either extraordinarily stable binding or mild competitive elution at room temperature. In order to further enhance the versatility of the ALFA system, we, here, aimed at developing an sdAb optimized for efficient elution at low temperatures. To achieve this, we followed a stringent selection scheme tailored to the specific application. We found candidates combining a fast capture of ALFA-tagged proteins with an efficient competitive elution at 4 °C in physiological buffer. Importantly, by employing a structure-guided semisynthetic library based on well-characterized NbALFA variants, the high specificity and consistent binding of proteins harboring ALFA-tags at either terminus could be maintained. ALFA SelectorCE, a resin presenting the cold-elutable NbALFACE, is an ideal tool for the one-step purification of sensitive protein complexes or temperature-labile enzymes. We believe that the general approach followed during the selection and screening can be transferred to other challenging sdAb discovery projects

Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives

The post-synaptic density protein 95 (PSD95) is a crucial scaffolding protein participating in the organization and regulation of synapses. PSD95 interacts with numerous molecules, including neurotransmitter receptors and ion channels. The functional dysregulation of PSD95 as well as its abundance and localization has been implicated with several neurological disorders, making it an attractive target for developing strategies able to monitor PSD95 accurately for diagnostics and therapeutics. This study characterizes a novel camelid single-domain antibody (nanobody) that binds strongly and with high specificity to rat, mouse, and human PSD95. This nanobody allows for more precise detection and quantification of PSD95 in various biological samples. We expect that the flexibility and unique performance of this thoroughly characterized affinity tool will help to further understand the role of PSD95 in normal and diseased neuronal synapses

RESOLFT Nanoscopy of Fixed Cells Using a Z-Domain Based Fusion Protein for Labelling

<div><p>RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment.</p></div

RESOLFT super-resolution image of an entire CV-1 cell.

<p>The cell was decorated with primary antibodies against α-tubulin and FLASR. Scale bar: 5 μm.</p

Immunolabelling with FLASR.

<p>(A) Schematic of FLASR (ZZ-rsEGFP2_tandem) bound to an immunoglobulin protein. (B-D) Maximum intensity projections of confocal z-stacks of methanol fixed mammalian CV-1 cells immunolabelled with antibodies against β-actin (B), vimentin (C) and α-tubulin (D). Subsequently, purified FLASR (red) was used to decorate the primary antibodies. Nuclei were stained with DAPI (blue). Scale bars: 50 μm.</p

RESOLFT nanoscopy of methanol fixed cells.

<p>Comparison of RESOLFT super-resolution microscopy and the corresponding confocal microscopy images of CV-1 cells decorated with primary antibodies against vimentin (A), α-tubulin (B) and the nuclear pore complex protein Nup153 (C). (D) Line-profiles of the fluorescence intensities recorded between the arrowheads in (A-C), as indicated (confocal: light blue; RESOLFT: red). The line profiles in (1–3) are averaged across five adjacent line profiles that were perpendicular across the respective filament. The distance between two adjacent line profiles was the edge length of one pixel. Scale bars: 1 μm.</p

Immunolabelling with an M-rsEGFP2_tandem fusion protein.

<p>Maximum intensity projections of confocal microscopy z-stacks of methanol fixed CV-1 cells immunolabelled with antibodies against β-actin (A) and vimentin (B). The purified recombinant fusion protein M-rsEGFP2_tandem was used to decorate the primary antibodies (red). Nuclei were labelled with DAPI (blue). Scale bars: 50 μm.</p

DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1. Pulse-chases combined with mutagenesis studies reveal that Ser1 strongly influences Syk stability. Furthermore, DPP9 silencing reduces Cbl interaction with Syk, suggesting that DPP9 processing is a prerequisite for Syk ubiquitination. Consistently, DPP9 inhibition stabilizes Syk, thereby modulating Syk signalling. Taken together, we demonstrate DPP9 as a negative regulator of Syk and conclude that DPP9 is a novel integral aminopeptidase of the N-end rule pathway. DOI: http://dx.doi.org/10.7554/eLife.16370.00