FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection

Protein Eng Des Sel. 2019 Dec 31;32(6):289-296. doi: 10.1093/protein/gzz047.

Abstract

FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.

Keywords: ED-B; PASylation; VEGFR-3; anticalin; eGFP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Green Fluorescent Proteins / genetics*
  • Lipocalins / chemistry
  • Lipocalins / genetics*
  • Lipocalins / metabolism*
  • Models, Molecular
  • Molecular Imaging / methods*
  • Protein Conformation
  • Protein Engineering*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / metabolism*

Substances

  • Lipocalins
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins