The use of mRNA display to select high-affinity protein-binding peptides

Abstract

We report the use of ''mRNA display,'' an in vitro selection technique, to identify peptide aptamers to a protein target. mRNA display allows for the preparation of polypeptide libraries with far greater complexity than is possible with phage display. Starting with a library of Ϸ10 13 random peptides, 20 different aptamers to streptavidin were obtained, with dissociation constants as low as 5 nM. These aptamers function without the aid of disulfide bridges or engineered scaffolds, yet possess affinities comparable to those for monoclonal antibodyantigen complexes. The aptamers bind streptavidin with three to four orders of magnitude higher affinity than those isolated previously by phage display from lower complexity libraries of shorter random peptides. Like previously isolated peptides, they contain an HPQ consensus motif. This study shows that, given sufficient length and diversity, high-affinity aptamers can be obtained even from random nonconstrained peptide libraries. By engineering structural constraints into these ultrahigh complexity peptide libraries, it may be possible to produce binding agents with subnanomolar binding constants.affinity tag ͉ peptide library ͉ peptide aptamer ͉ Strep-tag ͉ streptavidin

Site-specific selfish genes as tools for the control and genetic engineering of natural populations

“…First, further developments are needed in the re-targeting of these selfish genes to novel host sequences, though important progress has been made, both by design and by selection (Segal et al 1999;Guo et al 2000;Buchholz & Stewart 2001;Wilson et al 2001;Chevalier et al 2002;Santoro & Schultz 2002;Seligman et al 2002;Takahashi & Fujiwara 2002). Second, it must be demonstrated that these elements can be made to work in the prospective target species: insects, for example, are natural hosts only for the LINE-like elements.…”

A Handbook for DNA‐Encoded Chemistry

“…To date, display techniques employing such principles as phage display [7,8], ribosome display [9,10], yeast display [11], covalent display [12], mRNA display [13,14], and other conceptually analogous methodologies [15,16] profoundly impact the way novel drugs are discovered, yielding new and more efficacious classes of therapeutic agents (e.g., antibody drug conjugates) [17,18]. DNA-encoded library technology aims to extend the realm and the potential of these display approaches to the en masse interrogation of small synthetic organic molecules.…”

Optimisation of a multivalent Strep tag for protein detection

“…Each streptavidin monomer binds one biotin molecule with femtomolar affinity, one of the strongest non-covalent interactions observed in nature [8]. Over the past 15 years several groups have developed peptide binders to the tetrameric streptavidin protein [9][10][11][12][13] and a variety of protein engineering approaches have been applied to tailor affinity for specific applications [14][15][16]. This provides a simple and clean system for protein purification as a streptavidin binding peptide can be selectively eluted from a streptavidin affinity column using appropriate concentrations of biotin.…”

“…Consequently, several groups have set out to find higher affinity alternatives to the Strep-tags [9,13]. Using a mRNA display library,…”

“…Wilson and coworkers, for example, have identified a 38 amino acid long peptide (streptavidin-binding peptide, SBP), which binds with nanomolar affinity [13].…”