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Cloning, expression and characterization of human kininogen domain 3
Author(s) -
Auerswald Ennes A.,
Rössler Dieter,
Mentele Rainer,
Assfalg-Machleidt Irmgard
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)80628-8
Subject(s) - cloning (programming) , computational biology , domain (mathematical analysis) , characterization (materials science) , microbiology and biotechnology , kininogen , expression cloning , chemistry , biology , biochemistry , computer science , peptide sequence , gene , mathematics , materials science , kallikrein , nanotechnology , enzyme , mathematical analysis , programming language
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).