The paper “characterization and optimization of two-chain folding pathways of insulin via native chain assembly” in Communications Chemistry is here: https://go.nature.com/2I6LDsC
If we knew well about a long history of insulin preparation when we started the project of two-chain insulin folding several years ago, we did not put our foot in this difficult, but important research area. Indeed, we were lucky in that we could begin the project just out of our curiosity. At the time, our group in Tokai University was almost finishing characterization of oxidative folding pathways of various small single-chain proteins, such as ribonuclease A and lysozyme, by using dihydroxyselenolane oxide (DHSox), which is a highly reactive and selective water-soluble oxidant for disulfide (SS) formation developed in our laboratory. We were looking for our next step, when I met her.
Her name was Setsuko Ando, a lecturer of Fukuoka University at that time, who was interested in the synthesis and function of relaxin, a representative two-chain mammalian hormone having a similar SS-bond topology to insulin. She kindly provided us samples of the A- and B-chains obtained by solid-phase peptide synthesis (SPPS) in her laboratory. It was of our fortune that the native relaxin was generated, although the yield was not good (less than 10%) under standard oxidative protein folding conditions, just by mixing the A- and B-chains, having no protection on the amino acid side-chains, with DHSox. The result encouraged us strongly to promote our new project of insulin folding ahead.
After years, we have now elucidated the two-chain oxidative folding pathways of insulin clearly. The success has further enabled us to optimize the two-chain assembly conditions and thus establish the native chain assembly (NCA) protocol for insulin preparation,
in collaboration with researchers of Osaka University, Tohoku University, and Max Planck Institute of Biochemistry. With this protocol, human insulin and human type-2 relaxin can be obtained in up to 49% and 47% yields, respectively, just by mixing the A- and B-chains without applying any protections on the cysteine residues or a tether connecting the A- and B-chains. We hope that this simple NCA protocol will be useful for chemical synthesis of various foldable insulin analogs.
Michio Iwaoka and Kenta Arai