A Picomolar MBP-Binding Nanobody for Target Enrichment and Modular Complex Engineering

Engineering a Monomeric Nanopore Scaffold for Multiplexed Protein Detection.

Intracellular screening of nanobodies reveals an intrabody that inhibits ITCH E3 ubiquitin ligase

Nanobodies are a class of small, monomeric camelid antibody fragments that can bind target antigens with high affinity and specificity. Their small size, structural simplicity, and limited reliance on disulfide bonding makes them attractive for intracellular expression for labeling and perturbing cellular processes in live cells. However, screening campaigns carried out exclusively in vitro often yield antigen binders that fail to perform well in live cells due to low expression, misfolding, or mistargeting. We demonstrate that traditional in vitro screening of a nanobody library combined with an intracellular bioluminescence resonant energy transfer (BRET) proximity sensor approach for sequence down-selection can yield strong in vitro binders that also perform well as intrabodies, in this case capable of binding to, and inhibiting the enzymatic activity of, ITCH E3 ubiquitin ligase in human cells. This strategy allows a more direct and scalable path toward intrabody discovery.

Generation and evaluation of multimers of anti C-reactive protein Nb and alkaline phosphatase based on three self-assembly peptides

Nanobodies (Nbs) have been extensively utilized in medical diagnosis and therapies due to their advantages, such as ease of genetic manipulation and efficient soluble expression in prokaryotic systems. However, enhancing their binding affinity to antigens to match or even exceed that of polyclonal antibodies for high-sensitivity bioassays remains a challenge. Therefore, in the present study, a novel strategy to enhance the binding affinity of antibodies to antigens was developed by fusing an anti-C-reactive protein Nb (CRPNb) to the N-terminus of self-assembling peptides such as right-handed coiled coil [RHCC; derived from a right-handed coiled-coil peptide of an archaebacterium (Staphylothermus marinus)], verotoxin (VTB; the B-subunit of Escherichia coli verotoxin) and C4-binding protein (C4bp; derived from human plasma C4-binding protein α-chain). For functional detection, alkaline phosphatase (AP) was further fused to the C-terminus of RHCC, VTB and C4bp, respectively. This approach enabled the formation of CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers. These were then expressed as soluble cytoplasmic proteins in the E. coli strain BL21 (DE3) and purified by imidazole elution. The protein expression was assessed by western blotting. Additionally, the stability of the multimeric constructs was evaluated using a direct ELISA, whilst their sensitivity was assessed using a competitive ELISA. The CRPNb-VTB-AP pentamers and CRPNb-RHCC-AP tetramers demonstrated antigen-specific recognition and enhanced affinity compared with the CRP-AP monomer based on a direct ELISA. Furthermore, they exhibited good thermal stability based on a direct ELISA. At 80˚C, CRPNb-VTB-AP, CRPNb-AP and CRPNb-RHCC-AP retained ~85, 75 and 60% activity, while CRPNb-H only retained ~40%. After incubation at 80˚C for 55 min, the two multimers still maintained ~40% activity. The results suggested them to be suitable for storage and transportation at ambient temperatures. In conclusion, a novel nanobody-fusion protein platform was established in the present study. The fusion proteins functioned as high-affinity binders for target antigens and heat-stable signal tracers for immunoassay detection and quantitative analysis, providing a novel type of thermostable immunoreagent.

Profiling Protein‐Peptide Interactions by Yeast Surface Display

A protocol for discovering protein‐binding peptides using a very large, target‐agnostic yeast surface display library containing approximately 6.1 × 109 unique clones and providing broad coverage of short peptide sequence space is described.

Overcoming the Limitations of Protein A: Evolution of Bacterial Protein-Based and Synthetic Affinity Ligands for High-Performance IgG Purification

Monoclonal antibodies (mAbs) are widely used as therapeutic molecules for the treatment of serious diseases, primarily cancer. The market for mAbs is one of the fastest-growing segments of the biopharmaceuticals industry. Purification is a crucial stage in the production of mAbs. While staphylococcal protein A (SpA) affinity chromatography remains the gold standard in industrial mAb purification, its limitations—low alkaline stability and insufficient binding capacity of SpA, as well as the need for harsh acidic elution conditions—have driven extensive efforts for novel progressive affinity ligands. This review focuses on the development and performance of bacterial protein-based affinity resins for the purification of class G immunoglobulins (IgGs), including conventional proteins A and G, the promising protein L, and the more recently discovered protein M (from M. genitalium), each offering unique specificities for different antibody fragments and species. Hybrid ligands combining domains from multiple bacterial proteins are also discussed, along with next-generation synthetic alternatives such as affibodies, affimers, nanobodies, etc., as well as peptide-based or mixed-mode ligands. The key finding is that the reliable and time-tested resins like those based on protein A continue to dominate the market, although future trends also point toward smaller, more stable, and cost-effective synthetic ligands for specific applications.

Comprehensive Mapping of Immune Nanobody Repertoires with NanoMAP 2267451

Nanobodies have recently emerged as an alternative to classical antibodies in therapeutic and diagnostic contexts, promising improved stability and simpler manufacturing. However, many labs still rely on low throughput conventional screening methods for nanobody discovery. Here we report streamlined experimental and computational tools for discovery of nanobodies, permitting deep characterization of the binding properties of immune repertoires. To improve nanobody discovery, we developed NanoMAP, an integrated experimental and computational pipeline for nanobody discovery. We immunized alpacas with a pool of antigens, and created a phage display library from circulating B-cells. We then panned this phage display library on each antigen separately, and used competitors or antigen variants to assess complex binding phenotypes of the immune repertoire. Finally, we sequenced the panned libraries and developed a clustering method that allows data to be aggregated within B-cell clonal families, improving signal-to-noise ratios and reducing the complexity of the repertoire. We tested NanoMAP on three distinct pools of targets, collecting data on close to 1M unique nanobody sequences. We found that our specialized clustering method outperformed standard sequence clustering, producing clonal families that are coherent in sequence and function. By aggregating sequencing data within clonal families, NanoMAP produced reliable and rich data on binding phenotypes for each antigen. Using this information, we discovered nanobodies recognizing functionally relevant, and evolutionarily conserved sites on each antigen, demonstrating the broad utility of our methods. NIAID R01AI25704, NIGMS 5K12GM133314-07 Computational and Systems Immunology (COMP)