We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.

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This report describes a new cell-surface display system, the Secretion and Capture Technology (SECANT™) platform, which relies on in vivo biotinylation of the protein of interest followed by its capture on the avidinated surface of the parent cell. Cell sorting techniques are then used to isolate clones that display target-binding protein. A distinguishing feature of this method is its ability to display complex proteins, such as full-length immunoglobulin G (IgG) antibodies, on living cells. In this proof-of-concept study, Saccharomyces cerevisiae cells that displayed Herceptin IgG were isolated from a 10,000-fold excess of cells that displayed a lysozyme-binding antibody.