Nanocontainer derived from silkworm carotenoprotein for carotenoid extraction and presentation in biotechnology and biomedical applications

Found in many organisms, soluble carotenoproteins are considered as antioxidant nanocarriers for biomedical applications, although the structural basis for their carotenoid transfer function, a prerequisite for rational bioengineering, is largely unknown. We report crystal structures of the Carotenoid-Binding Protein from Bombyx mori (BmCBP) in apo- and zeaxanthin (ZEA)-bound forms. We use spectroscopy and calorimetry to characterize how ZEA and BmCBP mutually affect each other in the complex, identify key carotenoid-binding residues, confirm their roles by crystallography and carotenoid-binding capacity of BmCBP mutants and reconstitute BmCBP complexes with biomedically-relevant xanthophylls lutein, zeaxanthin, canthaxanthin and astaxanthin. By cost-effectively and scalably solubilizing xanthophylls from various crude herbal extracts, His-tagged BmCBP remains monomeric and forms a dynamic nanocontainer delivering carotenoids to liposomes and to other carotenoid-binding proteins, which in particular makes the Orange Carotenoid Protein, a promising optogenetic tool, photoactive. Furthermore, BmCBP(ZEA) administration stimulates fibroblast growth, which paves the way for its biomedical applications. ### Competing Interest Statement The authors have declared no competing interest. * AL : actinic light βCar : β-carotene ASESC : analytical size-exclusion spectrochromatography AstaP : astaxanthin-binding protein AXT : astaxanthin CAN : canthaxanthin CD : circular dichroism CTDH : C-terminal domain homolog DA : dark-adapted ECN : echinenone HOOP : hydrogen-out-of-plane IMAC : immobilized metalaffinity chromatography IPTG : isopropyl-β-thiogalactoside LA : light-adapted LED : lightemitting diode MEF : mouse embryonic fibroblasts OCP : orange carotenoid protein SAXS : small-angle X-ray scattering SDS-PAGE : sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEC : size-exclusion chromatography SEC-MALS : size-exclusion chromatography coupled to multi-angle light scattering START : steroidogenic acute regulatory lipid transfer protein TRITC : Tetramethylrhodamine isothiocyanate ZEA : zeaxanthin