SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

Abstract

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. We have recently developed an irreversible protein-peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide-peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide-peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture.bionanotechnology | synthetic biology | metastasis | antibody | nanobiotechnology

Programmable polyproteams built using twin peptide superglues

“…Polyproteams should be a powerful platform to dissect the spatial requirements for cellular signaling, such as in immunity and differentiation (51)(52)(53). Other applications of this simple route to new biological architectures may include vaccination (2), biomaterials (34,(54)(55)(56), multienzyme organization (9), and enhancing capture of circulating tumor cells (57).…”

Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging

“…Further, the amount of antibody required for molecular imaging is generally far lower than that of therapeutic requirements [42] reducing the chance for an immunogenic reaction. Fierer et al [43] and Siegmund et al [20] engineered the SpyTag/SpyCatcher system to ligate two peptides, SpyTag, and Ktag using a SpyLigase enzyme. This system provides another avenue to eliminate the immune response as the SpyCatcher is removed from the ligated product.…”

Recent Advances in Chemoenzymatic Bioconjugation Methods

“…63 The second-generation design permits for the ligation of two complementary peptide tags (11 and 13 residues, respectively) by an engineered SpyLigase enzyme. 64 The full potential of this technology remains to be explored.…”