Journal articles

Although multivalent binding to surfaces is an important tool in nanotechnology, quantitative information about the residual valency and orientation of surface-bound molecules is missing. To address these questions, we study streptavidin (SAv) binding to commonly used biotinylated surfaces such as supported lipid bilayers (SLBs) and self-assembled monolayers (SAMs).

Engineering modular platforms to control biomolecular architecture can advance both the understanding and the manipulation of biological systems. Icosahedral particles uniformly displaying single antigens stimulate potent immune activation and have been successful in various licensed vaccines. However, it remains challenging to display multiple antigens on a single particle and to induce broader immunity protective across strains or even against distinct diseases.

Gram-positive bacteria use reactive thioester-containing proteins to form covalent bonds, which may enable strong adhesion to host surfaces, but how these proteins selectively adhere to different surfaces is not clear. The Editors' Pick by Echelman et al. applied single-molecule force spectroscopy to show that an adhesin protein can regenerate its thioester in the absence of pulling. This selective interaction would represent a new principle of mechanical proof-reading, whereby only reactions supporting anchorage of the bacterium are maintained.

Vaccine development efforts have recently focused on enabling strong immune responses to poorly immunogenic antigens, via display on multimerisation scaffolds or virus like particles (VLPs). Typically such studies demonstrate improved antibody titer comparing monomeric and nano-arrayed antigen. There are many such studies and scaffold technologies, but minimal side-by-side evaluation of platforms for both the amount and efficacy of antibodies induced.

Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central research tool. Dye-NHS modification of streptavidin severely damaged ligand binding, necessitating development of a new streptavidin-retaining ultrastable binding after labeling.

Biological processes often depend on the harmonious interplay of multiple macromolecules. Biotechnology has had great success in applying and modifying individual components, but the building of multi-component teams is at an early stage. Cells are intelligent in sensing their environment, so manipulating just one signal can limit potency and promote side-effects for therapeutics. Here we critically assess the latest advances in irreversibly connecting individual protein units, through different spontaneous or catalysed reactions.

Simple, efficient reactions for connecting biological building-blocks open up many new possibilities. Here we have designed SnoopLigase, a protein that catalyzes site-specific transamidation, forming an isopeptide bond with more than 95% efficiency between two peptide tags, SnoopTagJr and DogTag. We initially developed these components by three-part splitting of the Streptococcus pneumoniae adhesin RrgA. The units were then engineered, guided by structure, bioinformatic analysis of sequence homology, and computational prediction of stability.

Vaccines based on virus-like particles (VLPs) can induce potent B cell responses. Some non-chimeric VLP-based vaccines are highly successful licensed products (e.g., hepatitis B surface antigen VLPs as a hepatitis B virus vaccine). Chimeric VLPs are designed to take advantage of the VLP framework by decorating the VLP with a different antigen. Despite decades of effort, there have been few licensed chimeric VLP vaccines. Classic approaches to create chimeric VLPs are either genetic fusion or chemical conjugation, using cross-linkers from lysine on the VLP to cysteine on the antigen.

Simple polymeric scaffolds have yielded dramatic effects on cell behavior. For more sophisticated phenotypes, precise and efficient chemistries are desired to incorporate proteins into these scaffolds. Here we derivatize hyaluronan with an elastin-like polypeptide containing telechelic SpyTags (HA-SpyTag). Our second network component, the TriCatcher protein, had two SpyCatchers and a terminal SnoopCatcher. Mixing HA-SpyTag with TriCatcher led to rapid hydrogel formation, via spontaneous amidation.