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In-vitro display technologies combine two important advantages for identifying and optimizing ligands by evolutionary strategies. First, by obviating the need to transform cells in order to generate and select libraries, they allow a much higher library diversity. Second, by including PCR as an integral step in the procedure, they make PCR-based mutagenesis(More)
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(More)
  • D. Lipovšek
  • 2011
Adnectins™ are a new family of therapeutic proteins based on the 10th fibronectin type III domain, and designed to bind with high affinity and specificity to therapeutically relevant targets. Adnectins share with antibody variable domains a beta-sheet sandwich fold with diversified loops, but differ from antibodies in primary sequence and have a simpler,(More)
Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic(More)
The 10th human fibronectin type III domain ((10)Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity (10)Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three(More)
We report a method for in vitro selection of catalytically active enzymes from large libraries of variants displayed on the surface of the yeast S. cerevisiae. Two libraries, each containing approximately 2 x 10(6) variants of horseradish peroxidase (HRP), were constructed; one involved error-prone PCR that sampled mutations throughout the coding sequence,(More)
The structural features determining efficient biosynthesis, stability in the membrane and, after solubilization, in detergents are not well understood for integral membrane proteins such as G protein-coupled receptors (GPCRs). Starting from the rat neurotensin receptor 1, a class A GPCR, we generated a separate library comprising all 64 codons for each(More)
The tenth human fibronectin type three domain ((10)Fn3) is a small (10 kDa), extremely stable and soluble protein with an immunoglobulin-like fold, but without cysteine residues. Selections from (10)Fn3-based libraries of proteins with randomized loops have yielded high-affinity, target-specific antibody mimics. However, little is known about the(More)
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(More)
The mRNA display approach to in vitro protein selection is based upon the puromycin-mediated formation of a covalent bond between an mRNA and its gene product. This technique can be used to identify peptide sequences involved in macromolecular recognition, including those identical or homologous to natural ligand epitopes. To demonstrate this approach, we(More)