Antibody-Linked Fluorogen-Activating Proteins for Antigen Detection and Cell Ablation.

We demonstrate selective labeling of cell surface proteins using fluorogen-activating proteins (FAPs) conjugated to standard immunoglobulins (IgGs). Conjugation was achieved with a polypeptide reagent comprised of an N-terminal photoactivatable Fc-binding domain and a C-terminal FAP domain. The resulting FAP-antibody conjugates were effective agents for protein detection and cell ablation in cultured mammalian cells and for visualizing cell-cell contacts using a tethered fluorogen assay. Because our approach allows FAP-antibody conjugates to be generated for most currently available IgGs, it should have broad utility for experimental and therapeutic applications.

Site-Specific Labeling of Cyanine and Porphyrin Dye-Stabilized Nanoemulsions with Affibodies for Cellular Targeting.

Recently, it has been shown that amphiphilic dyes such as Indocyanine Green (ICG) and Protoporphyrin IX (PpIX) can solubilize hydrophobic colloids and/or drugs by driving the formation of stable nanoemulsions. These nanoemulsions are unique in that they can be composed entirely of functional and clinically used materials; however, they lack bio-orthogonal chemical handles for the facile attachment of targeting ligands. The ability to target nanoparticles is desirable because it can lead to improved specificity and reduced side effects. Here, we describe variants of ICG and PpIX with azide handles that can be readily incorporated into dye-stabilized nanoemulsions and facilitate the attachment of targeting ligands via click-chemistry in a simple, scalable, and reproducible reaction. As a model system, an anti-Her2 affibody was site-specifically attached to both ICG and PpIX-stabilized nanoemulsions with encapsulated superparamagnetic iron oxide nanoparticles.

Ferritin Nanocages with Biologically Orthogonal Conjugation for Vascular Targeting and Imaging.

Genetic incorporation of biologically orthogonal functional groups into macromolecules has the potential to yield efficient, controlled, reproducible, site-specific conjugation of affinity ligands, contrast agents, or therapeutic cargoes. Here, we applied this approach to ferritin, a ubiquitous iron-storage protein that self-assembles into multimeric nanocages with remarkable stability, size uniformity (12 nm), and endogenous capacity for loading and transport of a variety of inorganic and organic cargoes. The unnatural amino acid, 4-azidophenylalanine (4-AzF), was incorporated at different sites in the human ferritin light chain (hFTL) to allow site-specific conjugation of alkyne-containing small molecules or affinity ligands to the exterior surface of the nanocage. The optimal positioning of the 4-AzF residue was evaluated by screening a library of variants for the efficiency of copper-free click conjugation. One of the engineered ferritins, hFTL-5X, was found to accommodate ∼14 small-molecule fluorophores (AlexaFluor 488) and 3-4 IgG molecules per nanocage. Intravascular injection in mice of radiolabeled hFTL-5X carrying antibody to cell adhesion molecule ICAM-1, but not control IgG, enabled specific targeting to the lung due to high basal expression of ICAM-1 (43.3 ± 6.99 vs 3.48 ± 0.14%ID/g for Ab vs IgG). Treatment of mice with endotoxin known to stimulate inflammatory ICAM-1 overexpression resulted in 2-fold enhancement of pulmonary targeting (84.4 ± 12.89 vs 43.3 ± 6.99%ID/g). Likewise, injection of fluorescent, ICAM-targeted hFTL-5X nanocages revealed the effect of endotoxin by enhancement of near-infrared signal, indicating potential utility of this approach for both vascular targeting and imaging.

Proximity-Based Sortase-Mediated Ligation.

Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site-specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity-based sortase-mediated ligation (PBSL), which improves the ligation efficiency to over 95 % by linking the target protein to SrtA using the SpyTag-SpyCatcher peptide-protein pair. By expressing the target protein with SpyTag C-terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher-SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step.

Multiple Antibiotic Resistance Plasmids Allow Scalable,PCR-Mediated DNA Manipulation and Near-Zero Background Cloning.

We have constructed two plasmids that can be used for cloning as templates for PCR- -based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418) and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.