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.

Keywords: antibody; bionanotechnology; metastasis; nanobiotechnology; synthetic biology.

Fig. 1.

Principle of SpyLigase design. (A) Reaction in CnaB2 domain between Lys and Asp with catalytic Glu to generate an isopeptide bond. (B) Splitting of the CnaB2 domain to give three modules. Cartoon of SpyTag peptide (blue), KTag peptide (pink), and SpyLigase (green) from CnaB2 domain (Protein Data Bank ID code 2X5P), with the residues involved in reaction colored black. (C) SpyLigase ligated SpyTag to KTag. SpyLigase was mixed with SpyTag-MBP and SUMO-KTag for 24 h before boiling and SDS/PAGE with Coomassie staining. SpyLigase EQ and SpyTag DA-MBP are nonreactive controls.

Fig. 2.

SpyLigase ligation characteristics. (A) SpyLigase ligated SpyTag and KTag at N- and C-termini. Tags were fused N-terminally on MBP or C-terminally on SUMO, incubated with SpyLigase for 24 h, and analyzed by SDS/PAGE with Coomassie staining. (B) SpyLigase ligated KTag to internal SpyTag sites, surrounding Zif268, analyzed after 24 h by SDS/PAGE with Coomassie staining. (C) Temperature dependence of ligation: SpyLigase was incubated with SpyTag-MBP and SUMO-KTag for 24 h at the indicated temperatures and ligation quantified from SDS/PAGE with Coomassie staining (mean of triplicate ±1 SD). (D) Electrospray MS shows SUMO-SpyTag linked to SUMO-KTag after incubation with SpyLigase, with loss of water.

Fig. 3.

Affibody polymerization with SpyLigase. (A) Cartoon of SpyLigase covalently joining KTag on one affibody to SpyTag on another, so directing polymerization. (B) Polymerization of anti-EGFR affibody for 24 h by SpyLigase, analyzed by SDS/PAGE with Coomassie staining. (C) Polymerization of anti-HER2 affibody for 48 h by SpyLigase, analyzed by SDS/PAGE with Coomassie staining.

Fig. 4.

Enhanced cell capture with affibody polymers. (A) Flow cytometry of EGFR expression in three cancer cell lines with the use of anti-EGFR antibody (+mAb, red) or no primary antibody control (no mAb, blue). (B) Recovery of high-EGFR MDA-MB-468 using magnetic beads coated with KTag-AffiEGFR-SpyTag affibody polymer (polymeric beads, blue) or affibody monomer (monomeric beads, red; mean of triplicate ±1 SD). (C) Recovery of low-EGFR BT474 or nonexpressing 721.221 cells as in B.