Open AccessThe epidermal growth factor-like domain from tissue plasminogen activator. Cloning in E. coli, purification and ESR studies of its interaction with human blood platelets.
Author(s) -
Tadeusz Pietrucha,
Wojciech J. Stec,
And̀rzej Okruszek,
Bogdan Uznański,
Maria Koziołkiewicz,
Andrzej Wilk,
Andrzej Płucienniczak,
Maria Świątkowska,
Czesław S. Cierniewski
Publication year - 1994
Publication title -
acta biochimica polonica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.452
H-Index - 78
eISSN - 1734-154X
pISSN - 0001-527X
DOI - 10.18388/abp.1994_4770
Subject(s) - chemistry , epidermal growth factor , microbiology and biotechnology , platelet , fusion protein , biochemistry , peptide , recombinant dna , activator (genetics) , biology , gene , receptor , immunology
To examine whether the epidermal growth factor (EGF)-like domain Pro47-Asp87 is involved in the interaction of tissue plasminogen activator (t-PA) with platelets, we have expressed this domain in E. coli. The peptide fragment was produced from a plasmid expression vector as a fusion protein with beta-galactosidase Met1-Val444 at high yield in eight clones of E. coli. The fusion protein was purified and subjected to mild acid hydrolysis with formic acid, then the peptide Pro47-Asp87, identified by immunoblotting using specific antibodies to t-PA, was isolated by HPLC. After incubation with blood platelets spin labelled with 16-doxylstearic acid or 5-doxylstearic acid, the Pro47-Asp87 peptide fragment reduced fluidity of the membrane lipid bilayer to the same extent as did intact t-PA as indicated by ESR measurements. Our data suggest that the EGF-like domain of t-PA can directly interact with blood platelets and thus it seems to contain those sites of the t-PA molecule that bind the platelet membrane components.