Interaction of cysteine proteinases with recombinant kininogen domain 2, expressed in Escherichia coli

FEBS Letters

Björk, Ingemar¹  Martin, Nancy C.²  Prasthofer, Thomas W.²  Ylinenjärvi, Karin¹ 

[1] Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Box 575, S-751 23 Uppsala, Sweden;Department of Medical and Physiological Chemistry, Uppsala University, Uppsala Biomedical Center, Box 575, S-751 23 Uppsala, Sweden

关键词: Kininogen;    Domain;    Cysteine proteinase;    Cysteine proteinase inhibitor;    Cystatin;    Bacterial expression;    AMC;    7-amido-4-methylcoumarin;    DTT;    dithiothreitol;    Omp A;    outer membrane protein A of E. coli;    PCR;    polymerase chain reaction;    Suc;    succinyl;    Z;    benzyloxycarbonyl;   

DOI  :  10.1016/0014-5793(94)01380-J

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The calpain-binding domain 2 of the kininogens, the major plasma inhibitors of cysteine proteinases, was expressed in Escherichia coli. Expression of soluble protein was optimal at 15°C and was augmented by growing the bacteria in sorbitol and betaine. The recombinant domain showed high affinity (K _i 0.3–1 nM) for cathepsin L and papain, and a somewhat lower affinity (K _i ∼ 15 nM) for calpain. The binding to cathepsin H was substantially weaker, and no inhibition of actinidin and cathepsin B was detected. The affinity for cathepsin L was comparable to that reported for the domain isolated from plasma L-kininogen, whereas the affinities for papain and calpain were about tenfold lower. The latter difference may be due to the recombinant domain being nonglycosylated.

【 授权许可】

Unknown