Cloning, expression and characterization of human kininogen domain 3

FEBS Letters

Assfalg-Machleidt, Irmgard¹  Auerswald, Ennes A.²  Mentele, Rainer²  Rössler, Dieter² 

[1] Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Ludwig-Maximilians-Universität München, Germany;Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität München, Germany

关键词: Kininogen;    Human;    Cysteine proteinase inhibitor;    Cystatin;    Periplasmic expression;    E. coli;    rKD3;    recombinant human kininogen domain 3 (ANSM-[G253-S377] kininogen);    PCR;    polymerase chain reaction;    IPTG;    isopropyl-β-d-thiogalactoside;   

DOI  :  10.1016/0014-5793(93)80628-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The internal domain 3 of the heavy chain of human kininogen, a cysteine proteinase inhibitor, was amplified by a polymerase chain reaction from the kininogen cDNA clone phKG36. The DNA fragment was expressed in Escherichia coli using the ompA expression vector pASK.40 and the resulting protein was isolated from periplasm, purified by S-carboxymethylpapain affinity- and ion-exchange chromatography. The recombinant human kininogen domain 3 is 92% pure, reacts with anti-kininogen antibodies and is actively inhibitory. The expected amino acid sequence of ANSM-[G253-S377] kininogen was confirmed; the inhibitor has a molecular mass of 14,396 Da and an isoelectric point of 6.0 (pH). The determined K _i values of the complexes with papain and cathepsin L are similar to those measured previously with proteolytically liberated kininogen domain 3, and those of single-domain cystatins, like chicken egg white cystatin. However, recombinant kininogen domain 3 is a weak inhibitor of cathepsin B (K _i = 63 nM) as it has been found for native L-kininogen (K _i = 340 nM).

【 授权许可】

Unknown