Expression, purification and characterization of the second Kunitz-type protease inhibitor domain of the human WFIKKN protein.
Recently we have described a novel secreted protein (the WFIKKN protein) that consists of multiple types of protease inhibitory modules, including two tandem Kunitz-type protease inhibitor-domains. On the basis of its homologies we have suggested that the WFIKKN protein is a multivalent protease inhibitor that may control the action of different proteases. In the present work we have expressed the second Kunitz-type protease inhibitor domain of the human protein WFIKKN in Escherichia coli, purified it by affinity chromatography on trypsin-Sepharose and its structure was characterized by CD spectroscopy. The recombinant protein was found to inhibit trypsin (Ki = 9.6 nm), but chymotrypsin, elastase, plasmin, pancreatic kallikrein, lung tryptase, plasma kallikrein, thrombin, urokinase or tissue plasminogen activator were not inhibited by the recombinant protein even at 1 microm concentration. In view of the marked trypsin-specificity of the inhibitor it is suggested that its physiological target may be trypsin.
Nagy
Alinda
A
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary.
Trexler
Mária
M
Patthy
László
L
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D011480:Protease Inhibitors; D011506:Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C424444:WFIKKN2 protein, human; D014357:Trypsin