Article info Vol. 2  No. 2   pp.  62 ~ 67
Title Improvement of crystal quality for X-ray diffraction of the problematic redox protein complex, thioredoxin and thioredoxininteracting protein
Authors Jungwon Hwang and Myung Hee Kim*
Institutions Infection and Immunity Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea. *Correspondence: mhk8n@kribb.re.kr
Abstract 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 redox-sensitive 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.