# Oxidative Folding of a Two‐Chain Protein Having Three Interchain Disulfide Bonds. Synthesis of Bromelain Inhibitor VI Through Native Chain Assembly

## TL;DR

Scientists successfully synthesized a complex two-chain protein using a simple folding method, achieving a high yield and full activity.

## Contribution

The study demonstrates the first successful synthesis of a two-chain protein with three interchain disulfide bonds using native chain assembly.

## Key findings

- Bromelain inhibitor VI was synthesized with a high yield of 53% using native chain assembly.
- The synthesized protein showed complete inhibitory activity against bromelain.
- The folding process's rate-limiting step involved intermediates with specific disulfide bond configurations.

## Abstract

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 (3SSH) and one‐SS intermediates of the L‐chain (1SSL). This achievement opens a door to the chemical synthesis of unprecedent multichain proteins with more complicated SS‐bond topologies.

Bromelain inhibitor VI, a two‐chain protein stabilized by three interchain SS bonds and two intrachain SS bonds, was synthesized in an incredibly high isolated yield (53%) just by mixing the two native chains in a buffer solution. This success made a record of oxidative protein folding for the most complicated and difficult one.