Protein Cages Engineered for Interaction-Driven Selective Encapsulation of Biomolecules.

Hollow protein cages have become attractive drug delivery vehicles with high biocompatibility and precise functional/structural manipulability. However, difficulties in effective cargo loading inside the cages have been limiting further development of protein cage-based drug carriers. Here, we developed a specific interaction-driven encapsulation and cellular delivery strategy for various biomolecules by engineering a porous protein cage. The computationally designed hyperstable mi3 protein cage was circularly permuted to fuse the cancer targeting RGD tripeptide to the cage surface and SpyTag (ST), which forms a covalent bond with SpyCatcher (SC), to the cage inner cavity. SC-fused proteins with different sizes and charges could be stably and actively encapsulated in the engineered nanocage via the ST/SC reaction. Cargo protein encapsulation inside the cage was directly confirmed by cryo-electron microscopy (EM) structure determination. In addition, SC-fused monomeric avidin was added to the nanocage to encapsulate various biotinylated (nonprotein) cargos such as oligonucleotides and the anticancer drug doxorubicin. All cargo molecules loaded onto the engineered mi3 were effectively delivered to cells. This work introduces a highly versatile cargo loading/delivery strategy, where loading/delivery interactions, cargo molecules, and cell targeting moieties can be further varied for optimal cellular drug delivery.

Specific capture, recovery and culture of cancer cells using oriented antibody-modified polystyrene chips coated with agarose film.

Agarose gel can be used for three dimensional (3D) cell culture because it prevents cell attachment. The dried agarose film coated on a culture plate also protected cell attachment and allowed 3D growth of cancer cells. We developed an efficient method for agarose film coating on an oxygen-plasma treated micropost polystyrene chip prepared by an injection molding process. The agarose film was modified to maleimide or Ni-NTA groups for covalent or cleavable attachment of photoactivatable Fc-specific antibody binding proteins (PFcBPs) via their N-terminal cysteine residues or 6xHis tag, respectively. The antibodies photocrosslinked onto the PFcBP-modified chips specifically captured the target cells without nonspecific binding, and the captured cells grew 3D modes on the chips. The captured cells on the cleavable antibody-modified chips were easily recovered by treatment of commercial trypsin-EDTA solution. Under fluidic conditions using an antibody-modified micropost chip, the cells were mainly captured on the micropost walls of the chip rather than on the bottom of it. The presented method will also be applicable for immobilization of oriented antibodies on various microfluidic chips with different structures.

Covalent and Oriented Surface Immobilization of Antibody Using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli.

Fc-specific antibody binding proteins (FcBPs) with the minimal domain of protein G are widely used for immobilization of well-oriented antibodies onto solid surfaces, but the noncovalently bound antibodies to FcBPs are unstable in sera containing large amounts of antibodies. Here we report novel photoactivatable FcBPs with photomethionine (pMet) expressed in E. coli, which induce Fc-specific photo-cross-linking with antibodies upon UV irradiation. Unfortunately, pMet did not support protein expression in the native E. coli system, and therefore we also developed an engineered methionyl tRNA synthetase (MRS5m). Coexpression of MRS5m proteins successfully induced photoactivatable FcBP overexpression in methionine-auxotroph E. coli cells. The photoactivatable FcBPs could be easily immobilized on beads and slides via their N-terminal cysteine residues and 6xHis tag. The antibodies photo-cross-linked onto the photoactivatable FcBP-beads were resistant from serum-antibody mediated dissociation and efficiently captured antigens in human sera. Furthermore, photo-cross-linked antibody arrays prepared using this system allowed sensitive detection of antigens in human sera by sandwich immunoassay. The photoactivatable FcBPs will be widely applicable for well-oriented antibody immobilization on various surfaces of microfluidic chips, glass slides, and nanobeads, which are required for development of sensitive immunosensors.