Synonymous Codons and Hydrophobicity Optimization of Post-translational Signal Peptide PelB Increase Phage Display Efficiency of DARPins

DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44- and 12-fold compared to PelBWT and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelBWT, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency

Genetically Encoded Protease Substrate Based on Lanthanide-Binding Peptide for Time-Gated Fluorescence Detection

The study of biomolecular interactions is at the heart of biomedical research. Fluorescence and Förster resonance energy transfer (FRET) are potent and versatile tools in studying these interactions. Fluorescent proteins enable genetic encoding which facilitates their use in recombinant protein and in vivo applications. To eliminate the autofluorescence background encountered in applications based on fluorescent proteins, lanthanide labels can be used as donor fluorophores. Their long emission lifetime enables the use of time-gating that significantly improves assay sensitivity. In this work, we have combined the favorable characteristics of a terbium-ion-containing lanthanide-binding peptide (Tb³⁺-LBP) and green fluorescent protein (GFP) in a FRET-based homogeneous protease activity assay. The used genetically engineered construct had LBP and GFP sequences at adjacent ends of a linker that encoded the recognition sequence for caspase-3. Caspase proteases are central mediators in apoptosis and, consequently, are of great interest in the pharmaceutical industry. The designed fluorogenic protease substrate was applied for the detection of caspase-3 activity. We were able to demonstrate, for the first time, the applicability of a Tb³⁺-LBP–GFP energy-transfer pair in a protease activity assay. The intrinsically fluorescent and genetically encodable components enable easy expression of the construct without the need of cumbersome chemical labeling. By varying the fluorescent protein and the protease specificity of the internal linker sequence, the method can be applied for the detection of a wide variety of proteases

Homogeneous Detection of Avidin Based on Switchable Lanthanide Luminescence

We have developed switchable lanthanide luminescence-based binary probe technology for homogeneous detection of avidin, which is a tetrameric protein. Two different nonluminescent label moietiesa light-absorbing antenna ligand and a lanthanide ion carrier chelatewere conjugated to separate biotins, which is known as avidin’s natural ligand. The assay was based on binding of the two differently labeled biotins on separate binding sites on the target protein and consequent self-assembly of a luminescent complex from the two label moieties. Specific luminescence signal was observed only at the presence of the target protein. The characteristics of the switchable lanthanide luminescence assay were compared to the reference assay, based on lanthanide resonance energy transfer. Both assays had a limit of detection in the low-picomolar concentration range; however, the lanthanide chelate complementation-based assay had wider dynamic range and its optimization was more straightforward. The switchable lanthanide luminescence technology could be further applied to generic protein detection, using reagents that are analogous to the proximity ligation assay principle

Broad-Spectrum Noncompetitive Immunocomplex Immunoassay for Cyanobacterial Peptide Hepatotoxins (Microcystins and Nodularins)

A broad-spectrum noncompetitive immunoassay allowing sensitive and simple detection of a group of similar compounds would be an ideal tool for screening low-molecular weight analytes (<2000 Da) having many variants. However, the development of an essential antibody pair capable of sandwich-type recognition of the analytes’ small generic core structure is a demanding task due to limited space available for simultaneous binding of two different antibodies. We report here a generic noncompetitive assay for cyanobacterial microcystins (MCs) and nodularins (Nod), a group of structurally related small cyclic peptides (∼1000 Da) with more than 100 naturally occurring analogs. The assay is based on the unique combination of a generic anti-immunocomplex (anti-IC) single-chain fragment of antibody variable domain (scFv) and a monoclonal antibody capable of binding to an Adda-group (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4­(<i>E</i>),6­(<i>E</i>)-dienoic acid) present in all MCs/Nod. The anti-IC scFv was isolated from a large synthetic antibody library with phage display and used to develop a single-step sandwich-type noncompetitive immunocomplex assay. The sensitive time-resolved immunofluorometry-based assay is capable of detecting all the 11 tested commonly occurring hepatotoxins (MC-LR, -dmlR, -RR, -dmRR, -LA, -LY, -LF, -LW, -YR, -WR, and Nod-R) at concentration below 0.1 μg/L in a 1 h assay. Using MC-LR, the most studied toxic and widely distributed of the toxins, the calculated detection limits (based on blank + 3SD response) are ∼0.026 μg/L in 1 h and ∼0.1 μg/L in 10 min assay time. This is by far the fastest reported immunoassay for MCs and Nod with a detection limit far below the World Health Organization’s guideline limit (1 μg/L of MC-LR equivalent in drinking water). The assay was validated with spiked tap and lake water as well as with environmental surface water samples. The developed assay provides a simple, rapid, and highly sensitive tool for the quantitative detection of MCs/Nod with the additional benefit of automation and high-throughput possibilities for large scale screening of drinking and environmental surface water samples. Furthermore, the study describes the first demonstration of the assay intended for the detection of an analyte group comprising similar low-molecular weight compounds exhibiting the benefits of a reagent excess type assay

GTP-Specific Fab Fragment-Based GTPase Activity Assay

GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2′/3′-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4^GTP) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations