Unfolding of Recombinant
Single-chain Insulin in Denaturants Containing Thiol Reagents
GUO Zhan-Yun, QIAO Zhi-Song, FENG You-Min*
( State Key Laboratory of Molecular Biology, Institute of Biochemistry and
Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy
of Sciences, Shanghai 200031, China )
Abstract Recombinant
single�–hain insulin (PIP) contains three disulfide bonds. In the presence of
denaturants and thiol reagents, the native structure of PIP was disturbed and
its native disulfides were shuffled to form a mixture of scrambled isomers
which have different degrees of unfolding. In this paper the unfolding degrees
of PIP in urea or guanidine hydrochloride containing 0.2 mmol/L 2-mercaptoethanol
was analyzed by reverse-phase HPLC and far-UV circular dichroism(CD). The
peptide mapping of PIP scrambles demonstrated that PIP had shuffled its native
disulfides under the condition we used. Among others, a major non-natural PIP
disulfide isomer was purified and its refolding in vitro was
investigated. These results show that PIP has only one thermodynamically stable
disulfide linkage, and the non-natural disulfide isomers could refold in
vitro efficiently to from native PIP. On basis of these, the differences
between PIP, IGF-I and insulin on unfolding and refolding were discussed.
Key words recombinant single�–hain insulin； denaturation； unfolding； disulfide； refolding
*Corresponding author： Tel, 86-21-64374430； Fax, 86-21-64338357； e-mail, [email protected]