ABSTRACT
The ability to deliver small protein scaffolds intracellularly could enable the targeting and inhibition of many therapeutic targets that are not currently amenable to inhibition with small-molecule drugs. Here, we report the engineering of small protein scaffolds with anionic polypeptides (ApPs) to promote electrostatic interactions with positively charged nonviral lipid-based delivery systems. Proteins fused with ApPs are either complexed with off-the-shelf cationic lipids or encapsulated within ionizable lipid nanoparticles for highly efficient cytosolic delivery (up to 90%). The delivery of protein inhibitors is used to inhibit two common proto-oncogenes, Ras and Myc, in two cancer cell lines. This report demonstrates the feasibility of combining minimally engineered small protein scaffolds with tractable nanocarriers to inhibit intracellular proteins that are generally considered "undruggable" with current small molecule drugs and biologics.
Subject(s)
Nanoparticles , Neoplasms , Cytosol , Humans , Liposomes/chemistry , Nanoparticles/chemistryABSTRACT
Antibodies can be developed to directly inhibit almost any protein, but their inability to enter the cytosol limits inhibitory antibodies to membrane-associated or extracellular targets. Developing a cytosolic antibody delivery system would offer unique opportunities to directly inhibit and study intracellular protein function. Here we demonstrate that IgG antibodies that are conjugated with anionic polypeptides (ApPs) can be complexed with cationic lipids originally designed for nucleic acid delivery through electrostatic interactions, enabling close to 90% cytosolic delivery efficiency with only 500 nM IgG. The ApP is fused to a small photoreactive antibody-binding domain (pAbBD) that can be site-specifically photocrosslinked to nearly all off-the-shelf IgGs, enabling easy exchange of cargo IgGs. We show that cytosolically delivered IgGs can inhibit the drug efflux pump multidrug resistance-associated protein 1 (MRP1) and the transcription factor NFκB. This work establishes an approach for using existing antibody collections to modulate intracellular protein function.
Subject(s)
Cytosol/metabolism , Immunoglobulin G/metabolism , Biological Transport , Cell Line , Cell Membrane/metabolism , HEK293 Cells , Humans , Immunoglobulin G/chemistry , Lipid Metabolism , Models, Biological , Multidrug Resistance-Associated Proteins , NF-kappa B/antagonists & inhibitors , Static ElectricityABSTRACT
S. aureus sortase A (SrtA), a calcium-dependent transpeptidase, is frequently employed to site-specifically label the C-terminus of recombinant proteins bearing an LPXTG SrtA recognition motif. Unfortunately, SrtA suffers from low turnover rates, resulting in poor ligation efficiencies even with optimized reaction conditions. In this chapter, we describe proximity-based sortase-mediated ligation (PBSL), which uses the SpyTag-SpyCatcher peptide-protein pair to link SrtA to target proteins and dramatically improves reaction rate and ligation efficiency.
Subject(s)
Aminoacyltransferases/chemistry , Bacterial Proteins/chemistry , Cysteine Endopeptidases/chemistry , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins , Staining and Labeling , Aminoacyltransferases/genetics , Bacterial Proteins/genetics , Cloning, Molecular , Cysteine Endopeptidases/genetics , Gene ExpressionABSTRACT
S. aureus sortase A (SrtA), a calcium-dependent bacterial transpeptidase, is commonly used to site-specifically label proteins containing a LPXTG SrtA recognition motif with a wide array of chemical moieties. A major limitation of sortase-mediated labeling, however, is SrtA's poor binding affinity to its recognition motif, resulting in long reaction times and poor ligation efficiencies. Here we describe proximity-based sortase-mediated ligation (PBSL), which utilizes the SpyTag-SpyCatcher peptide-protein pair to tether target proteins with a SrtA recognition motif to SrtA, dramatically increasing their local concentrations and overcoming this limitation.
Subject(s)
Aminoacyltransferases/chemistry , Bacterial Proteins/chemistry , Cysteine Endopeptidases/chemistry , Peptides/chemistry , Staphylococcus aureus/enzymologyABSTRACT
Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortaseâ 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.
