Enzymatic properties of phage-displayed fragments of human plasminogen.
Two low-molecular-mass forms of human plasminogen, plasminogen-(543-791)-peptide (micro-plasminogen), comprising the serine protease domain, and plasminogen-(444-791)-peptide (mini-plasminogen), which in addition contains kringle 5, were displayed on filamentous phage by fusion to the N-terminus of the minor coat protein pIII, to levels of 0.5 molecules micro-plasminogen-pIII/phage particle and 0.1 molecules mini-plasminogen-pIII/phage particle. The proenzymes, quantitatively activated by urokinase, showed catalytic efficiencies that were virtually identical to their soluble counterparts, and activity remained associated with the phage as demonstrated by phage ELISA and biopanning with human alpha2-antiplasmin or the inhibitor Phe-Pro-Arg-CH2Cl. Micro-plasminogen-pIII was activated by streptokinase and staphylokinase, two non-enzymatic plasminogen activators, to the same extent as by urokinase. Activated forms of mini-plasminogen-pIII micro-plasminogen-pIII and mini-plasminogen dissolved 125I-labelled fibrin films in a dose-dependent time-dependent manner, with 50% lysis in 20 h requiring 0.52, 3.2 and 0.46 nM active plasmin, respectively. Thus, proenzyme moieties derived from plasminogen can be successfully displayed on phage with maintenance of their enzymatic properties. The micro-plasminogen and mini-plasminogen phage-display systems may be useful to study mechanisms of plasminogen activation.
Lasters
I
I
Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Belgium.
Van Herzeele
N
N
Lijnen
H R
HR
Collen
D
D
Jespers
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005342:Fibrinolysis / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D017912:Inovirus / Q000235:genetics; D007700:Kinetics; D008666:Metalloendopeptidases / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013300:Streptokinase / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology
D017931:DNA Primers; D010446:Peptide Fragments; C044190:miniplasminogen; D010958:Plasminogen; D004247:DNA; D013300:Streptokinase; D014568:Urokinase-Type Plasminogen Activator; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus