The epidermal growth factor-like domain from tissue plasminogen activator. Cloning in E. coli, purification and ESR studies of its interaction with human blood platelets

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 bet...

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Veröffentlicht in:Acta biochimica polonica 1994, Vol.41 (1), p.25-34
Hauptverfasser: Pietrucha, T, Stec, W J, Okruszek, A, Uznański, B, Koziołkiewicz, M, Wilk, A, Płucienniczak, A, Swiatkowska, M, Cierniewski, C S
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Sprache:eng
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Zusammenfassung: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.
ISSN:0001-527X
1734-154X
DOI:10.18388/abp.1994_4770