A Bifunctional Protein G-Streptavidin Fusion Protein for Antibody Detection and Circulating Tumor Cell Enrichment.

Abstract

Circulating tumor cells (CTCs) are important biomarkers of cancer metastasis; however, their extremely low abundance in the bloodstream poses significant challenges for sensitive and specific detection. Bifunctional fusion proteins that combine an IgG-binding domain with a signal-amplifying module offer a promising strategy to address this challenge. In this study, we constructed a recombinant bifunctional fusion protein, designated r-Protein G-SA, by fusing four tandem repeats of the high-affinity C3 domain of streptococcal Protein G to core streptavidin via a flexible (GGGGS)3 linker. The purified protein was obtained in high purity as assessed by SDS-PAGE and adopted a well-defined secondary structure as determined by circular dichroism (CD) spectroscopy. Surface plasmon resonance (SPR) analysis revealed a high apparent binding affinity for human IgG, with an apparent Kd of 13.98 nM. In immunofluorescence assays, the fusion protein enabled clear discrimination among SKBR3 (HER2 3+), ZR-75-1 (HER2 2+), and MCF-7 (HER2 1+) cells, whereas no detectable signal was observed in HER2-negative MDA-MB-231 and white blood cells (WBCs). In spiked rare-cell capture experiments (5, 50, 500, 1,000, or 2,000 SKBR3 cells spiked into 10 mL blood), recovery rates exceeded 80% with good linearity (R2 = 0.9994). Collectively, the r-Protein G-SA fusion protein retains both IgG Fc-binding and biotin-binding activities, enabling sensitive and specific labeling of target cells. These results demonstrate its potential as a versatile tool for antibody-based detection and rare-cell enrichment. Nevertheless, validation in clinical patient samples is required prior to any clinical application.