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) p rotein 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 comb inatorial immunoglobulin repertoire cloning, for routing of PAI-1 to t he periplasm and subsequent exposure on the surface of filamentous pha ges. Phage-displayed PAI-1 specifically binds to immobilized polyclona l and monoclonal anti-human PAI-1 antibodies. In addition, PAI-I retai ns its capacity to form equimolar complexes with its target serine pro tease tissue-type plasminogen activator (t-PA), as well as its ability to inhibit t-PA activity. Finally, we have explored and manipulated t he error-prone property of TaqI DNA polymerase during PCR amplificatio n of the full-length PAI-1 cDNA to generate a large library of predomi nantly single, random PAI-I mutants. In addition, a computer simulatio n program has been devised that converts the number of mutations per c odogenic region (in this case PAI-1) into actual mutant proteins. The PAI-1-phage mutant library is composed of 46% single and 34% double mu tants and 20% wild-type PAI-1 and can be employed to isolate mutants d efective in interactions of PAI-I with other components. The method de scribed here is applicable to other studies on the structure-function analysis of eukaryotic proteins.