A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.

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Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited β-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.The aggregation of proteins into amyloid fibers is associated with a growing list of diseases, including diabetes, Alzheimer's, Parkinson's, Huntington's and the prion diseases. In these disorders, proteins aggregate into long, unbranched fibers after misfolding into a β-sheet-rich conformation 1 . Though there are no approved therapies targeting amyloid formation directly, many organic molecules inhibit fibrillization in vitro [2][3][4][5][6][7] . Some, such as the chelator clioquinol (1), even have activity in vivo 4 . These results have inspired the hope of therapeutic applications for some molecules 3-5 . Curiously, many fibrillization inhibitors resemble molecules known to form promiscuous chemical aggregates. These colloidal particles are composed of small organic molecules and range in size from 50 to over 600 nm 8 . Once formed, they physically sequester proteins and inhibit enzymes nonspecifically 8,9 . Like many inhibitors of amyloid polymerization, these colloidal inhibitors are typically highly conjugated, hydrophobic and dye-like (Supplementary Table 1 online) 8,9 . A good example is the amyloid inhibitor Congo red (2), a dye that was one of the first molecules observed to exhibit colloidal inhibition 8 . The flavonoid baicalein (3), an inhibitor of α-synuclein polymerization 6 , resembles the known chemical aggregator quercetin (4), and 4,5-dianilinophthalimide (DAPH, 5), an inhibitor of Alzheimer's amyloid formation 2 , resembles the aggregator bisindoylmaleimide (6 ; Supplementary Fig. 1 online).Given that chemical aggregates function through enzyme sequestration, we wondered whether they might also sequester protein molecules from each other, thereby preventing amyloid polymerization. Here, we investigate this hypothesis in two classic amyloid-forming proteins: the yeast prion protein Sup35 (ref. 10 ) and the recombinant mouse prion protein recMoPrP 89-230 (ref. 11 ). We ask whether known chemical aggregators can inhibit amyloid fiber formation, whether known fibrillization inhibitors form colloidal aggregates and whether amyloid inhibition by these molecules is in fact mediated via colloidal aggregation.Eight known chemical aggregators and two known nonaggregators 8,9 were tested for inhibition of Sup35 fibrillization in a thioflavin T (ThT, 7) fluorescence assay. All eight inhibited Sup35 fibrillization b...

A conformational isoform of the mammalian prion protein (PrP Sc ) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-␤ diffraction: meridional intensity at 4.8 Å resolution, indicating the presence of ␤ strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four ␤-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brainderived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 Å meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 Å, absent from brain-derived prions, and indicating the presence of stacked ␤-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.amyloid ͉ protein ͉ neurodegeneration ͉ PrP ͉ ␤-helix