Orthogonal crosslinking of monobodies using a genetically encoded β-lactam amino acid

Nature uses disulfide bonds and some unusual inter-strand chemical crosslinks to rigidify protein structure and achieve specific function. Inspired by Nature, protein scientists have employed genetic code expansion technology to introduce latent electrophilic amino acids into protein structure for specific crosslinking with nearby nucleophilic residues via the proximity-driven reactions. Herein, we describe the experimental protocol for recombinant production of orthogonal crosslinked monobodies afforded by a genetically encoded β-lactam-lysine (BeLaK) and their characterization by gel electrophoresis and mass spectrometry. When introduced to the N-terminal β-strand of a series of supercharged monobodies, BeLaK enables efficient inter-strand crosslinking with a proximal lysine on a neighboring β-strand. Compared to its non-crosslinked counterpart, a BeLaK-crosslinked, +18-charged monobody showed higher thermal stability and greater cell permeability. The discovery of this BeLaK-crosslinked, rigidified immunoglobulin fold should facilitate the design of cell-permeable monobodies as potential protein-based therapies targeting the intracellular signaling proteins.