Chemical Synthesis and Chaperone Peptide Mediated Folding of Human Nerve Growth Factor by Expressed KAHA Ligation

Nerve growth factor (NGF) is a powerful neurotrophic protein for treating central nervous system diseases, but its therapeutic utility is limited by severe side effects, including hyperalgesia. These adverse effects arise from pleitropic receptor binding that can, in principle, be modulated by side chain mutations or modificationa task suited for chemical protein synthesis. Despite its small size (13 kDa), the chemical synthesis of NGF has been stymied by exceptional hydrophobicity and the requirement for a 104-residue N-terminal "chaperone peptide" for folding. This study presents a chemical synthesis of NGF using α-ketoacid-hydroxylamine (KAHA) ligations, featuring recombinant production of the chaperone peptide and its chemoselective conversion to a C-terminal α-ketoacid. A novel solubility tag, SOLACE, and ester-forming KAHA ligations enabled assembly of linear proNGF from three synthetic and one recombinant segment. Controlled folding and disulfide-bond formation mediated by the chaperone peptide followed by proteolytic cleavage yielded biologically active synthetic NGF as its noncovalent dimer. The synthetic NGF exhibited comparable activity to recombinant NGF in axon growth assays, establishing a platform for engineering NGF variants with tailored therapeutic properties. This approach provides a versatile framework for the semisynthesis of neurotrophins and related proteins that also require long chaperone peptides for proper folding.