Improvement of crystal quality for X-ray diffraction of the problematic redox protein complex, thioredoxin and thioredoxininteracting protein

Thioredoxin-interacting protein (TXNIP) regulates many biological processes by interacting with thioredoxin (TRX) in a redox-dependent fashion. Thus, elucidation of the mechanism, by which these two proteins interact, is a key to understand redox-dependent cell signaling. Recently, the TXNIP-TRX complex structure and their interacting mechanism have been published. Both TRX (containing 5 cysteine residues) and TXNIP (containing 11 cysteine residues) are highly redoxsensitive proteins, and are therefore extremely difficult to handle in vitro. Here, we present details of how these problematic redox proteins can be expressed, purified, and crystallized to a suitable quality for X-ray diffraction. Both proteins were expressed as a soluble complex in the E. coli Rosetta-gami (DE3) system in which disulfide bonds can form owing to trxB/gor mutation. Moreover, catching TXNIP in an intermediate state, in which TRX was bound, was crucial to obtain stable complex proteins linking to crystallization.