Journal articles

Cell isolation via antibody-targeted magnetic beads is a powerful tool for research and clinical applications, most recently for isolating circulating tumor cells (CTC). Nonetheless fundamental features of the cell-bead interface are still unknown. Here we apply a clinically relevant antibody against the cancer target HER2 (ErbB2) for magnetic cell isolation. We investigate how many target proteins per cell are sufficient for a cell to be isolated.

SpyTag is a peptide that spontaneously forms an amide bond with its protein partner SpyCatcher. SpyTag was fused at the N terminus of β-lactamase and SpyCatcher at the C terminus so that the partners could react to lock together the termini of the enzyme. The wild-type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized β-lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored.

SpyTag is a short peptide that forms an isopeptide bond upon encountering its protein partner SpyCatcher. This covalent peptide interaction is a simple and powerful tool for bioconjugation and extending what protein architectures are accessible. Here we review the origin and mechanism of SpyTag/SpyCatcher, focusing on recent innovative applications. Ligation of targeting-antibody with antigen provided a simple route to vaccine generation. SpyRings, from head-to-tail cyclisation, gave major enhancements in enzyme resilience.

Antibody affinity limits sensitivity of detection in many areas of biology and medicine. High affinity usually depends on achieving the optimal combination of the natural 20 amino acids in the antibody binding site. Here, we investigate the effect on recognition of protein targets of placing an unnatural electrophile adjacent to the target binding site. We positioned a weak electrophile, acrylamide, near the binding site between an affibody, a non-immunoglobulin binding scaffold, and its protein target.

Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, DNA amplification and nanotechnology. We identified a mutant streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. FtsK, a motor protein important in chromosome segregation, rapidly displaced streptavidin from biotinylated DNA, whereas traptavidin resisted displacement, indicating the force generated by Ftsk translocation.

FtsK translocates dsDNA directionally at >5 kb/s, even under strong forces. In vivo, the action of FtsK at the bacterial division septum is required to complete the final stages of chromosome unlinking and segregation. Despite the availability of translocase structures, the mechanism by which ATP hydrolysis is coupled to DNA translocation is not understood. Here, we use covalently linked translocase subunits to gain insight into the DNA translocation mechanism.

The interaction between SA (streptavidin) and biotin is one of the strongest non-covalent interactions in Nature. SA is a widely used tool and a paradigm for protein-ligand interactions. We previously developed a SA mutant, termed Tr (traptavidin), possessing a 10-fold lower off-rate for biotin, with increased mechanical and thermal stability. In the present study, we determined the crystal structures of apo-Tr and biotin-Tr at 1.5 Å resolution. In apo-SA the loop (L3/4), near biotin's valeryl tail, is typically disordered and open, but closes upon biotin binding.

Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequence-programmed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein.

Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions.