Jennifer Dias

[11] Cell-free synthesis of peptide libraries displayed on polysomes

Publisher Summary The polysome display system is a recent biological method for screening peptide libraries. Unlike the cell-based methods, polysome screening relies on the in vitro synthesis and amplification of the peptide sequences during each round of screening. This offers the potential of screening larger libraries and a more diverse collection of sequences since few biological biases should affect gene expression in vitro . A notable difference between the phage display and the current polysome display system is the affinities of the recovered peptides. It is found that high-affinity sequences (Kd values of less than 200 nM) predominate the pools of enriched sequences that suggests that polysome display may be monovalent. This is in contrast to the multivalent phage and plasmid display systems that can recover ligands, with affinities as low as 100μM. Monovalent peptide display limits the range of ligand families that are recovered, but is very effective in selecting higher affinity sequences, especially if mutagenic PCR 22 is incorporated between the rounds of selection. Repeated rounds of polysome screening result in certain sequences, dominating the population, and often the most frequently occurring sequence will bind to the target with the highest affinity.

Expression of cre recombinase as a reporter of signal transduction in mammalian cells.

BACKGROUND Cell-based reporter assays, which rely on a reporter gene under the control of a regulated promoter, are widely used to screen chemical libraries for novel receptor ligands. Here, we describe a reporter system that is based on ligand-induced DNA recombination to express the reporter gene. This system converts a transient activation of a signal transduction pathway into an amplified, constitutive and heritable expression of the reporter gene. RESULTS We constructed gene fusions of Cre recombinase and mammalian promoters regulated by calcium, nuclear receptors or cyclic AMP. Reporter systems, comprising a Cre gene fusion and a loxP/reporter gene, were used to study the kinetics and dose responses to compounds that activate or inhibit the corresponding signal transduction pathway. We compared these reporters with conventional reporter systems in which the reporter gene is under the direct control of the responsive promoter. Reporter gene expression of the Cre reporters was greater than that of conventional reporters and could be measured more than a week after adding the stimulus. For all pathways studied here, the dose responses of the Cre reporters are nearly identical to those of conventional reporter systems. CONCLUSIONS We have shown that Cre recombinase can be regulated by a variety of signal transduction pathways. It should therefore be possible to use receptor ligands to induce phenotypic conversion of mammalian cells for use in a variety of applications. One such application is high-throughput screening, and we developed loxP/luciferase reporter genes that provide an amplified and sustained luminescent response.