Journal articles

Virus-like particles (VLPs) can play important roles in prevention and therapy for infectious diseases and cancer. Here we describe recent advances in rational construction of VLP assemblies, as well as new approaches to enhance long-lasting antibody and CD8+ T cell responses. DNA origami and computational protein design identified optimal spacing of antigens. Chemical biology advances enabled simple and irreversible VLP decoration with protein or polysaccharide antigens.

The Neisseria meningitidis protein FrpC contains a self-processing module (SPM) undergoing autoproteolysis via an aspartic anhydride. Herein, we establish NeissLock, using a binding protein genetically fused to SPM. Upon calcium triggering of SPM, the anhydride at the C-terminus of the binding protein allows nucleophilic attack by its target protein, ligating the complex. We establish a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We optimize NeissLock using the Ornithine Decarboxylase/Antizyme complex.

Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses.

Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach.

Proteins span an extraordinary range of shapes, sizes and functionalities. Therefore generic approaches are needed to overcome this diversity and stream-line protein analysis or application. Here we review SpyTag technology, now used in hundreds of publications or patents, and its potential for detecting and controlling protein behaviour. SpyTag forms a spontaneous and irreversible isopeptide bond upon binding its protein partner SpyCatcher, where both parts are genetically-encoded.

Much of life's complexity depends upon contacts between proteins with precise affinity and specificity. The successful application of engineered proteins often depends on high-stability binding to their target. In recent years, various approaches have enabled proteins to form irreversible covalent interactions with protein targets. However, the rate of such reactions is a major limitation to their use. Infinite affinity refers to the ideal where such covalent interaction occurs at the diffusion limit.

We describe a method to generate monovalent quantum dots (QDs) using agarose gel electrophoresis. We passivated QDs with a carboxy-terminated polyethylene-glycol ligand, yielding particles with half the diameter of commercial QDs, which we conjugated to a single copy of a high-affinity targeting moiety (monovalent streptavidin or antibody to carcinoembryonic antigen) to label cell-surface proteins. The small size improved access of QD-labeled glutamate receptors to neuronal synapses, and monovalency prevented EphA3 tyrosine kinase activation.

Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization.

We present a methodology for targeting quantum dots to specific proteins on living cells in two steps. In the first step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches 10-bromodecanoic acid onto a 13 amino acid recognition sequence that is genetically fused to a protein of interest. In the second step, quantum dots derivatized with HaloTag, a modified haloalkane dehalogenase, react with the ligated bromodecanoic acid to form a covalent adduct.

Biotechnology is often limited by weak interactions. We suggest that an ideal interaction between proteins would be covalent, specific, require addition of only a peptide tag to the protein of interest, and form under a wide range of conditions. Here we summarize peptide tags that are able to form spontaneous amide bonds, based on harnessing reactions of adhesion proteins from the bacterium Streptococcus pyogenes. These include the irreversible peptide-protein interaction of SpyTag with SpyCatcher, as well as irreversible peptide-peptide interactions via SpyLigase.