Ferritin Conjugates With Multiple Clickable Amino Acids Encoded by C-Terminal Engineered Pyrrolysyl-tRNA Synthetase

Abstract

This study reports the application of expanding genetic codes in developing protein cage-based delivery systems. The evolved Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)•tRNAPyl pairs derived from directed evolution are examined to probe their recognition for para-substituted phenylalanine analogs. The evolved MmPylRS, AzFRS, harboring a wide range of substrates, is further engineered at the C-terminal region into another variant, AzFRS-MS. AzFRS-MS shows suppression of the elevated sfGFP protein amount up to 10 TAG stop codons when charging p-azido-l-phenylalanine (AzF, 4), which allows the occurrence of click chemistry. Since protein nanocages used as drug delivery systems that encompass multiple drugs through a site-specific loading approach remain largely unexplored, as a proof of concept, the application of AzFRS-MS for the site-specific incorporation of AzF on human heavy chain ferritin (Ftn) is developed. The Ftn-4 conjugate is shown to be able to load multiple fluorescence dyes or a therapeutic agent, doxorubicin (Dox), through the strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction. Aiming to selectively target Her2+ breast cancer cells, Ftn-4-DOX conjugates fused with a HER2 receptor recognition peptide, anti-Her2/neu peptide (AHNP), is developed and demonstrated to be able to deliver Dox into the cell and to prolong the drug release. This work presents another application of evolved MmPylRS systems, whose potential in developing a variety of protein conjugates is noteworthy.

Simple plant‐based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor‐targeted drug delivery and bioimaging in cancer therapy

“…Sun et al, 2021; X. Zhang et al, 2019) that can be tested in PCPs. More advanced modifications would allow selective binding to cells other than those presenting TfR1 (Wang et al, 2021). Additionally, drug‐loaded ferritin particles can be coupled to probes such as cell membrane‐binding peptides (Wang et al, 2021) via N‐terminal SNAP‐tag fusions, allowing their rapid deployment as targeted theranostics (Padayachee et al, 2019).…”

“…More advanced modifications would allow selective binding to cells other than those presenting TfR1 (Wang et al, 2021). Additionally, drug‐loaded ferritin particles can be coupled to probes such as cell membrane‐binding peptides (Wang et al, 2021) via N‐terminal SNAP‐tag fusions, allowing their rapid deployment as targeted theranostics (Padayachee et al, 2019). Drug‐loaded ferritin nanocarriers are highly versatile, and the low‐cost production method presented here could be expanded to supply researchers working on tumor‐targeted drug delivery nanoformulations with the necessary quantities of protein.…”

Ferritin nanocages as efficient nanocarriers and promising platforms for COVID-19 and other vaccines development

“…Similarly, the inner surface of the ferritin nanocage may be engineered by genetically fusing hydrophobic peptides to the C-terminal end of ferritin subunits to increase absorption of insoluble, hydrophobic cargo [ 111 , 112 ]. Introducing cysteine residues to the interior of the nanocage has been a widely used approach to facilitate covalent attachment of dye molecules, drugs, or other active compounds through disulfide bonds [ 113 ] or to enable specific chemical reactions such as copper-free click chemistry [ 114 ].…”

Engineering Self-Assembling Protein Nanoparticles for Therapeutic Delivery

“…The interior nanoparticle surface is commonly modified to encapsulate specific materials (Figure E). It has been widely demonstrated that mutating interior-facing surface residues to hold a net charge facilitates electrostatic-mediated cargo encapsulation (e.g., encapsulation of supercharged fluorescent proteins or nucleic acids) (Figure E). ,, Interior surface residues can also be mutated to include side chains capable of specific chemical reactions like copper-free click chemistry . Alternatively, larger domains like affinity peptides can be seamlessly integrated into the protein nanoparticle interior through genetic fusion.…”

“…18,55,83 Interior surface residues can also be mutated to include side chains capable of specific chemical reactions like copper-free click chemistry. 84 Alternatively, larger domains like affinity peptides can be seamlessly integrated into the protein nanoparticle interior through genetic fusion. Genetic fusion of functional domains to the nanoparticle interior surface can either be bioactive themselves or enable specific cargo encapsulation such as mRNA and siRNA sequences.…”