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Abstract Domain antibodies such as monobodies provide an attractive immunoglobin fold for evolving high‐affinity protein binders targeting the intracellular proteins implicated in cell signalling. However, it remains a challenge to endow cell permeability to these small and versatile protein binders. Here, we report a streamlined approach combining orthogonal crosslinking afforded by a genetically encodedβ‐lactam‐lysine (BeLaK) and genetic supercharging to generate cell‐penetrating monobodies. When introduced to the N‐terminalβ‐strand of a series of supercharged monobodies, BeLaK enabled efficient inter‐strand crosslinking with the neighbouring lysine. Compared to its non‐crosslinked counterpart, a BeLaK‐crosslinked, +18‐charged monobody exhibited enhanced thermostability and greater cellular uptake at 40 nM. Moreover, this structurally rigidified, supercharged monobody inhibited ERK1/2 phosphorylation in KYSE‐520 esophageal cancer cell line at sub‐micromolar concentration, indicating significant endosomal escape after endocytosis. Together, the discovery of this BeLaK‐encoded, rigidified immunoglobin fold should facilitate the design of cell‐penetrating monobodies targeting intracellular signalling proteins. 

Abstract Compared to the disulfide bond, other naturally occurring intramolecular crosslinks have received little attention, presumably due to their rarity in the vast protein space. Here we presented examples of natural non‐disulfide crosslinks, which we refer to as orthogonal crosslinks, emphasizing their effect on protein topology and function. We summarize recent efforts on expanding orthogonal crosslinks by using either the enzymes that catalyze protein circularization or the genetic code expansion strategy to add electrophilic amino acids site‐specifically in proteins. The advantages and disadvantages of each method are discussed, along with their applications to generate novel protein topology and function. In particular, we highlight our recent work on spontaneous orthogonal crosslinking, in which a carbamate‐based crosslink was generated in situ, and its applications in designing orthogonally crosslinked domain antibodies with their topology‐mimicking bacterial adhesins.