tailieunhanh - Báo cáo khoa học: 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 prote-ase 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 multi-valent 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 inEscherichia coli, purified it by affinity chromatography on trypsin-Sepharose and its structure was characterized by CD spectroscopy | Eur. J. Biochem. 270 2101-2107 2003 FEBS 2003 doi Expression purification and characterization of the second Kunitz-type protease inhibitor domain of the human WFIKKN protein Alinda Nagy Maria Trexler and László Patthy Institute of Enzymology Biological Research Center Hungarian Academy of Sciences Budapest Hungary 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 K 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 M concentration. In view of the marked trypsin-specificity of the inhibitor it is suggested that its physiological target may be trypsin. Keywords Kunitz-domain multidomain protease inhibitor serine proteinases trypsin. Recently we have identified two closely related human proteins WFIKKN and WFIKKNRP each of which contain a WAP-domain a Follistatin Kazal domain an Immunoglobulin-domain two Kunitz-domains and an NTR-domain 2 3 . The tissue expression pattern of the two proteins however is markedly different suggesting that they have distinct biological roles. Whereas the WFIKKNRP gene is expressed primarily in ovary testis and brain the most significant expression of the WFIKKN gene is observed in adult pancreas liver and thymus. In view of the presence of WAP- Kazal- .