Oxidative Folding of a Two-Chain Protein Having Three Interchain Disulfide Bonds. Synthesis of Bromelain Inhibitor VI...

The recently renovated two-chain folding method, in which two native peptide chains without any sidechain protections and interchain scaffolds are just mixed in a buffer solution under optimized conditions, called native chain assembly (NCA), enabled efficient chemical synthesis of α-helix-rich insulin and its analogs, which are stabilized by two interchain disulfide (SS) bridges. Herein, this simple folding method has been successfully applied to the folding of a different-type two-chain protein, that is, bromelain inhibitor VI (BI-VI), which has abundant β-sheet structures and is stabilized by three interchain SS bridges. When the chemically synthesized native heavy (H)- and light (L)-chains of BI-VI were mixed at 4 °C in a pH of 10.0 buffer solution containing 2 mM GSH and 0.4 mM GSSG, native BI-VI was obtained surprisingly in a high isolated yield (53%) after 2 weeks. The obtained BI-VI showed complete inhibitory activity against bromelain, whereas each component chain exhibited essentially non-activity. The rate-limiting step of the two-chain folding was assumed to be the chain coupling between three-SS intermediates of the H-chain (3SS^H) and one-SS intermediates of the L-chain (1SS^L). This achievement opens a door to the chemical synthesis of unprecedent multichain proteins with more complicated SS-bond topologies.