Native chemical ligation enables stepwise assembly of proteins via unprotected ligation at cysteine.1 Total chemical synthesis of proteins is limited by the need to strategically place cysteines for native chemical ligation with possible subsequent desulfurization. In special cases where different protein fragments self-assemble, the resulting high effective concentration allows direct aminolysis of the thioester, enabling ligation without a cysteine.2 We have applied this principle to the synthesis of a 92-amino acid b-sheet monobody protein based on the tenth fibronectin type III domain. This required the combination of two separate strategies. First, the insoluble N-terminal portion was selectively modified by arginine substitution to enhance solubility.3 Second, the disconnection was chosen to permit self-assembly, maximizing high local concentration, and permitting rapid, direct aminolysis. The resulting protein retained binding to its original target as measured by ITC.