An in vitro polysome display system for identifying ligands from very large peptide libraries.

Abstract

We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 1012 random decapeptides was incubated in an Escherichia coil S30 coupled transcription/translation system. Polysomes were isolated and screened by afflnity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B. The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection. A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA. After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody. Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM. The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems.Peptide libraries derived from the cloning and expression of random-sequence oligonucleotides provide a rich source of ligands. These libraries, which often contain >108 recombinants, are characterized by the physical linkage of each peptide to its encoding DNA. This feature permits the affinity purification of peptides and associated DNA on an immobilized receptor and the DNA is sequenced to identify the peptide. This general approach provides a powerful tool for discovering ligands.A widely used version of this strategy is the display of peptides on the outer surface of filamentous phage particles. The peptide sequences are encoded by random oligonucleotides inserted into the 5' region of the genes encoding the capsid proteins pIII or pVIII (1-3). The peptides are expressed on the phage fused to the N terminus of the coat proteins. Several rounds of screening and amplification result in the enrichment of phage expressing peptides that bind to the receptor. Another system employs peptides fused to the C terminus of the lac repressor Lacd (4). The repressor protein physically links the peptides to the plasmid encoding them by binding to the lac operator sequences on the plasmid. The peptide-LacI-plasmid complexes are screened in a manner analogous to the phage system. These library approaches to ligand discovery often rely on the generation of huge numbers of peptides with the expectation that those of the appropriate structure will be rare. The size of a cell-based library is limited by the number of recombinants recovered from the transformation step. To create a peptide display system that avoids this limitation and is capable of screening much larger libraries, we developed the in vitro polysome system describe...

An Oriented Peptide Array Library (OPAL) Strategy to Study Protein-Protein Interactions

“…The display libraries employ a variety of matrices for individual peptide presentation, including pin (5), phage (6 -8), beads (9), DNA binding proteins (10), or ribosomes (11). Following multiple rounds of affinity enrichment steps, the identities of the selected peptides can be ascertained by DNA or peptide sequencing (4).…”

Beyond the Natural Proteome

“…It was the first in vitro selection method, inspired by work of Mattheakis and co-workers (Mattheakis, Bhatt & Dower, 1994) who demonstrated affinity selection using polysomes which enable the critical link between genotype and phenotype that is essential for any biopanning application. As an in vitro method ribosome display is not limited by transformation efficiency and can therefore be used to screen very large libraries.…”

“…They displayed a library of short, 13-mer opioid peptides on E. coli S30 followed by affinity screening against immobilized antibodies. Because the expressed proteins were coupled with mRNA, selected hits could be reverse transcribed into cDNA and then sequenced to determine the nucleotide sequence encoding the displayed peptide (Mattheakis et al, 1994).…”

Potential of phage-displayed peptide library technology to identify functional targeting peptides

“…These include synthetic one-bead-one-compound libraries [10][11][12], cell-free translation systems, such as mRNA display (peptide linked to mRNA by puromycin) [13][14][15], DNA display (DNA and peptide captured in compartments of water-in-oil emulsions) [16] and ribosome display libraries (peptide linked to mRNA by stalling the ribosome) [17][18][19], and biologic libraries, such as bacteria display [20][21][22], adenovirus display [23,24], baculovirus display [24] and bacteriophage display libraries [25][26][27][28]. GP Smith reported in 1985 that a bacteriophage could be manipulated to display foreign amino acid sequences on the surface of the phage by fusion to phage coat proteins without losing the ability of phage infectivity [28].…”