Random PCR Mutagenesis Screening of Secreted Proteins by Direct Expression in Mammalian Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS 1992-06, Vol.89 (12), p.5467-5471
• Main Authors: Rice, Glenn C., Goeddel, David V., Cachianes, George, Woronicz, John, Chen, Ellson Y., Williams, Steven R., Leung, David W.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Antibodies, Monoclonal; Biological and medical sciences; Biotechnology; Blood Proteins - genetics; Carcinoma, Hepatocellular; CD55 Antigens; Cell Line; Cells; Cellular biology; Deoxyribonucleic acid; DNA; DNA - genetics; Epitopes; Epitopes - analysis; Eukaryotes; expression; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Gene Library; Genetic technics; Hep G2 cells; Humans; Libraries; Liver Neoplasms; Mammals; Membrane proteins; Membrane Proteins - genetics; Membrane Proteins - metabolism; Methods. Procedures. Technologies; Models, Molecular; Mutagenesis; Mutagenesis, Site-Directed; Mutagenicity tests; Mutant screening; plasminogen activator; polymerase chain reaction; Polymerase Chain Reaction - methods; Protein Conformation; Receptors; Recombinant Fusion Proteins - metabolism; Restriction Mapping; screening; Tissue Plasminogen Activator - genetics; Tissue Plasminogen Activator - metabolism; Transfection; X-Ray Diffraction
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.12.5467

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.