Selective protein unfolding: a universal mechanism of action for the development of irreversible inhibitors.

High-throughput differential scanning fluorimetry of GFP-tagged proteins (HT-DSF-GTP) was applied for the identification of novel enzyme inhibitors acting by a mechanism termed: selective protein unfolding (SPU). Four different protein targets were interrogated with the same library to identify target-selective hits. Several hits selectively destabilized bacterial biotin protein ligase. Structure-activity relationship data confirmed a structure-dependent mechanism of protein unfolding. Simvastatin and altenusin were confirmed to irreversibly inactivate biotin protein ligase. The principle of SPU combined with HT-DSF-GTP affords an invaluable and innovative workflow for the identification of new inhibitors with potential applications as antimicrobials and other biocides.

A universal immuno-PCR platform for comparative and ultrasensitive quantification of dual affinity-tagged proteins in complex matrices.

Protein detection in complex biological fluids and matrices has become a widely diversified field utilizing a number of different technologies. The quantification of target proteins in complex media such as serum remains a challenge for most technologies such as mass spectrometry, ELISA and western blot. Quantitative Immuno-PCR has been heavily used for antigen detection in immunoassays, but minimally so for quantifying affinity-tagged proteins expressed or circulating in complex matrices--despite its high sensitivity and robustness--because it suffers from detrimental background effects arising from its extreme detection power. We report the development of a universal qIPCR-based platform for the reproducible detection of dual affinity-tagged protein analytes in crude complex matrices such as serum and cell culture media or lysates. The system uses a couple of high-affinity antibodies against two affinity tags (GFP and HA) for the detection of dual-tagged proteins. The dual-tagged analyte is immuno-captured by one of its tags, while the second tag is bound by a detection device consisting of a new kind of self-assembled antibody-DNA conjugate. The new qIPCR platform enabled picomolar quantification of dual-tagged sortase in crude serum in 4 h including the PCR step.

IgG-detection devices for the Tus-Ter-lock immuno-PCR diagnostic platform.

The number of new Immuno-PCR technologies and applications is steadily growing as a result of a general need for more sensitive immunoassays for early detection of diseases. Although Immuno-PCR has been demonstrated to be superior to its immunoassay counterpart, it is still regarded as a challenging technology due to various problems arising from its increased detection power, such as high background noise as well as substantial batch-to-batch reproducibility issues. Current efforts have intensified to produce homogeneous universal protein-DNA conjugates to simplify this technology and render it more robust. We have recently developed a new quantitative Immuno-PCR (qIPCR) technology using the Tus-Ter-lock (TT-lock) interaction to produce homogeneous protein-DNA conjugates that can detect very small numbers of disease-related antibodies. We now report the further development of the TT-lock Immuno-PCR platform for the quasi universal quantitative detection of antigens and mammalian IgG. For this, Tus was fused to various IgG-binding proteins--i.e. protein G, protein L and their LG chimera--and self-assembled to the TT-lock-T template. These detection devices were then evaluated and applied in various direct and indirect Immuno-PCR formats. The direct TT-lock qIPCR could detect goat anti-GFP IgG at concentrations as low as 0.3 pM and total human IgG in serum samples with great sensitivity. Further indirect TT-lock qIPCR systems were developed that could detect 1 pM of GFP and 10 pM of measles nucleoprotein. In all cases, the superiority of the TT-lock Immuno-PCR was demonstrated in terms of sensitivity over an analogous Protein G-Peroxidase ELISA.

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.