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 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.
Department of Biophysics, Medical University of Lódź, Poland.
T
T
Pietrucha
W J
WJ
Stec
A
A
Okruszek
B
B
Uznański
M
M
Koziołkiewicz
A
A
Wilk
A
A
Płucienniczak
M
M
Swiatkowska
C S
CS
Cierniewski
D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism*; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004815:Epidermal Growth Factor / Q000097:blood / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator / Q000737:chemistry*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D010959:Tissue Plasminogen Activator