Phage ESCape: an emulsion-based approach for the selection of recombinant phage display antibodies.

Antibody phage display technology is a well established method for selecting specific antibodies against desired targets. Although phage display is the most widely used method of generating synthetic antibodies, it is laborious to perform multiple selections with different antigens simultaneously using conventional manual methods. We have developed a novel approach to the identification and isolation of cells secreting phage encoding desirable antibodies that utilizes compartmentalization and Fluorescence Activated Cell Sorting (FACS). This method, termed Phage Emulsion, Secretion, and Capture (ESCape), allows us to individually query each phage against the antigen. Here, we demonstrate the ability of Phage ESCape to identify novel scFvs against a phosphopeptide epitope of the Her2 kinase from a phage display library containing approximately 10(8) synthetically diversified antibodies. Clones were analyzed by monoclonal phage ELISA against the Her2 phosphopeptide, and positive binders were identified as those showing a signal greater than 3-fold higher than the background signal against an irrelevant antigen. We isolated antibodies recognizing the phosphopeptide in a single round of selection by Phage ESCape, but the strength and specificity of the hits was substantially improved when the library was pre-enriched by a single round of biopanning. By minimizing the selection rounds required for phage display and using a FACS machine as a 'colony picker' equivalent, Phage ESCape has the potential to dramatically increase the throughput of in vitro screening methods.

Dynamic and complex transcription factor binding during an inducible response in yeast.

Complex biological processes are often regulated, at least in part, by the binding of transcription factors to their targets. Recently, considerable effort has been made to analyze the binding of relevant factors to the suite of targets they regulate, thereby generating a regulatory circuit map. However, for most studies the dynamics of binding have not been analyzed, and thus the temporal order of events and mechanisms by which this occurs are poorly understood. We globally analyzed in detail the temporal order of binding of several key factors involved in the salt response of yeast to their target genes. Analysis of Yap4 and Sko1 binding to their target genes revealed multiple temporal classes of binding patterns: (1) constant binding, (2) rapid induction, (3) slow induction, and (4) transient induction. These results demonstrate that individual transcription factors can have multiple binding patterns and help define the different types of temporal binding patterns used in eukaryotic gene regulation. To investigate these binding patterns further, we also analyzed the binding of seven other key transcription factors implicated in osmotic regulation, including Hot1, Msn1, Msn2, Msn4, Skn7, and Yap6, and found significant coassociation among the different factors at their gene targets. Moreover, the binding of several key factors was correlated with distinct classes of Yap4- and Sko1-binding patterns and with distinct types of genes. Gene expression studies revealed association of Yap4, Sko1, and other transcription factor-binding patterns with different gene expression patterns. The integration and analysis of binding and expression information reveals a complex dynamic and hierarchical circuit in which specific combinations of transcription factors target distinct sets of genes at discrete times to coordinate a rapid and important biological response.

Biochemical and genetic analysis of the yeast proteome with a movable ORF collection.

Functional analysis of the proteome is an essential part of genomic research. To facilitate different proteomic approaches, a MORF (moveable ORF) library of 5854 yeast expression plasmids was constructed, each expressing a sequence-verified ORF as a C-terminal ORF fusion protein, under regulated control. Analysis of 5573 MORFs demonstrates that nearly all verified ORFs are expressed, suggests the authenticity of 48 ORFs characterized as dubious, and implicates specific processes including cytoskeletal organization and transcriptional control in growth inhibition caused by overexpression. Global analysis of glycosylated proteins identifies 109 new confirmed N-linked and 345 candidate glycoproteins, nearly doubling the known yeast glycome.

Loss of ypk1 function causes rapamycin sensitivity, inhibition of translation initiation and synthetic lethality in 14-3-3-deficient yeast.

14-3-3 proteins bind to phosphorylated proteins and regulate a variety of cellular activities as effectors of serine/threonine phosphorylation. To define processes requiring 14-3-3 function in yeast, mutants with increased sensitivity to reduced 14-3-3 protein levels were identified by synthetic lethal screening. One mutation was found to be allelic to YPK1, which encodes a Ser/Thr protein kinase. Loss of Ypk function causes hypersensitivity to rapamycin, similar to 14-3-3 mutations and other mutations affecting the TOR signaling pathway in yeast. Similar to treatment with rapamycin, loss of Ypk function disrupted translation, at least in part by causing depletion of eIF4G, a central adaptor protein required for cap-dependent mRNA translation initiation. In addition, Ypk1 as well as eIF4G protein levels were rapidly depleted upon nitrogen starvation, but not during glucose starvation, even though both conditions inhibit translation initiation. These results suggest that Ypk regulates translation initiation in response to nutrient signals, either through the TOR pathway or in a functionally related pathway parallel to TOR.