RANDOM PCR MUTAGENESIS SCREENING OF SECRETED PROTEINS BY DIRECT EXPRESSION IN MAMMALIAN-CELLS

We have developed a general method for screening randomly mutagenized expression libraries in mammalian cells by using fluorescence-activated cell sorting (FACS). The cDNA sequence of a secreted protein is randomly mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA is inserted into an expression vector encoding the membrane glycophospholipid anchor sequence of decay-accelerating factor (DAF) fused to the C terminus of the secreted protein. This results in expression of the protein on the cell surface in transiently transfected mammalian cells, which can then be screened by FACS. This method was used to isolate mutants in the kringle 1 (K1) domain of tissue plasminogen activator (t-PA) that would no longer be recognized by a specific monoclonal antibody (mAb387) that inhibits binding of t-PA to its clearance receptor. DNA sequence analysis of the mutants and localization of the mutated residues on a three-dimensional model of the K1 domain identified three key discontinuous amino acid residues that are essential for mAb387 binding. Mutants with changes in any of these three residues were found to have reduced binding to the t-PA receptor on human hepatoma HepG2 cells but to retain full clot lysis activity.