Functional display of human plasminogen-activator inhibitor 1 (PAI-1) on phages: novel perspectives for structure-function analysis by error-prone DNA synthesis.
The synthesis of the human plasminogen-activator inhibitor 1 (PAI-1) protein in the cytoplasm of transformed Escherichia coli cells results in inactive protein preparations that can be activated by denaturation and renaturation. We have used the phagemid pComb3, designed for combinatorial immunoglobulin repertoire cloning, for routing of PAI-1 to the periplasm and subsequent exposure on the surface of filamentous phages. Phage-displayed PAI-1 specifically binds to immobilized polyclonal and monoclonal anti-human PAI-1 antibodies. In addition, PAI-1 retains its capacity to form equimolar complexes with its target serine protease tissue-type plasminogen activator (t-PA), as well as its ability to inhibit t-PA activity. Finally, we have explored and manipulated the error-prone property of TaqI DNA polymerase during PCR amplification of the full-length PAI-1 cDNA to generate a large library of predominantly single, random PAI-1 mutants. In addition, a computer simulation program has been devised that converts the number of mutations per codogenic region (in this case PAI-1) into actual mutant proteins. The PAI-1-phage mutant library is composed of 46% single and 34% double mutants and 20% wild-type PAI-1 and can be employed to isolate mutants defective in interactions of PAI-1 with other components. The method described here is applicable to other studies on the structure-function analysis of eukaryotic proteins.
Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Department of Molecular Biology, Amsterdam.
H
H
Pannekoek
M
M
van Meijer
R R
RR
Schleef
D J
DJ
Loskutoff
C F
CF
Barbas
D000911:Antibodies, Monoclonal; D003001:Cloning, Molecular / Q000379:methods*; D003090:Coliphages / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D016296:Mutagenesis; D010957:Plasmids; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis*
D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D004247:DNA