Design and screening of M13 phage display cDNA libraries

The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF) libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS) will be presented.     

The discovery of small-molecule mimicking peptides through phage display

Using peptides to achieve the functional and structural mimicry of small-molecules, especially those with biological activity or clear biotechnological applications, has great potential in overcoming difficulties associated with synthesis, or unfavorable physical properties. Combinatorial techniques like phage display can aid in the discovery of these peptides even if their mechanism of mimicry is not rationally obvious.The major focus of this field has been limited to developing biotin and sugar mimetics. However, the full "mimicry" of these peptides has not yet been fully established as some bind to the target with a different mechanism than that of the natural ligand and some do not share all of the natural ligand's binding partners. In this article, mimicry of small-molecules by phage display-discovered peptides is reviewed and their potential in biochemical and medical applications is analyzed.     

Comparison of bacterial and phage display peptide libraries in search of target-binding motif

Genetic engineering allows modification of bacterial and bacteriophage genes, which code for surface proteins, enabling display of random peptides on the surface of these microbial vectors. Biologic peptide libraries thus formed are used for high-throughput screening of clones bearing peptides with high affinity for target proteins. There are reports of many successful affinity selections performed with phage display libraries and substantially fewer cases describing the use of bacterial display systems. In theory, bacterial display has some advantages over phage display, but the two systems have never been experimentally compared. We tested both techniques in selecting streptavidin-binding peptides from two commercially available libraries. Under similar conditions, selection of phage-displayed peptides to model protein streptavidin proved convincingly better.     

The powerful combination of phage surface display of cDNA libraries and high throughput screening

Phage surface display of cDNA libraries facilitates cloning, expression and rapid selection of functional gene products physically linked to their genetic information through gene product-ligand interactions. Efficient screening technologies based on selective enrichment of clones expressing desired gene products allows, within a short time, the isolation of all ligand-specific clones that are present in a library. Manual identification of clones by restriction analysis and random sequencing is unlike to be successful for the isolation of gene products derived from rare mRNA species resulting from selection of the libraries using polyvalent ligands like serum from patients. Here we describe rapid handling of large numbers of individual clones selected from molecular libraries displayed on phage surface using the power of robotics-based high throughput screening. The potential of the combination of cDNA-phage surface display, with selection for specific interactions by functional screening and robotic technology is illustrated by the isolation of more sequences potentially encoding IgE-binding proteins than postulated from Western blot analyses using extracts derived from raw material of complex allergenic sources. The subsequent application of functional enrichment and robotics-based screening will facilitate the rapid generation of information about the repertoire of protein structures involved in allergic diseases.     

Two-dimensional diversity: screening human cDNA phage display libraries with a random diversity probe for the display cloning of phosphotyrosine binding domains

A random phosphopeptide probe (bio-pYZZZ) has been used for the isolation and identification of multiple SH2 domains from human cDNA-displaying phage libraries. In addition, on-phage analysis and quantification of binding affinities for these phage-displayed proteins has shown them to be functional domains, retaining the same characteristics as in their native state.     

EF-Tu binding peptides identified,dissected, and affinity optimized by phage display

The highly abundant GTP binding protein elongation factor Tu (EF-Tu) fulfills multiple roles in bacterial protein biosynthesis. Phage-displayed peptides with high affinity for EF-Tu were selected from a library of approximately 4.7 x 10(11) different peptides. The lack of sequence homology among the identified EF-Tu ligands demonstrates promiscuous peptide binding by EF-Tu. Homolog shotgun scanning of an EF-Tu ligand was used to dissect peptide molecular recognition by EF-Tu. All homolog shotgun scanning selectants bound to EF-Tu with higher affinity than the starting ligand. Thus, homolog shotgun scanning can simultaneously optimize binding affinity and rapidly provide detailed structure activity relationships for multiple side chains of a polypeptide ligand. The reported peptide ligands do not compete for binding to EF-Tu with various antibiotic EF-Tu inhibitors, and could identify an EF-Tu peptide binding site distinct from the antibiotic inhibitory sites.     

Predicting in vivo protein peptide interactions with random phage display

Binding sites in protein complexes occasionally map to small peptides within one or more proteins. Random peptide display methods simulate binding interactions by providing all possible peptide combinations with an equal opportunity to bind a protein of interest. The natural substrates for the protein are typically known in advance. However, it is often the case that such substrates are identified as putative partner proteins by using in vivo methods such as yeast two hybrid screening. Unfortunately, such methods often produce lengthy datasets of protein sequences and offer little mechanistic insight into how such interactions might take place in vivo. Here, we review an approach that addresses this problem. First, sequence alignment tools identify and characterize blocks of conserved sequences among peptides recovered during random peptide display. Next, searching programs detect similar blocks of conserved sequences within naturally occurring proteins to predict partner proteins. Finally, the significance of an interaction is tested using site specific mutagenesis, binding competition or co-immunoprecipitation experiments. This strategy should become increasingly powerful with the growing popularity of interaction studies, sequencing projects and microarray analyses in modern biology.     

Protein ligand design: from phage display to synthetic protein epitope mimetics in human antibody Fc-binding peptidomimetics

Phage display is a powerful method for selecting peptides with novel binding functions. Synthetic peptidomimetic chemistry is a powerful tool for creating structural diversity in ligands as a means to establish structure-activity relationships. Here we illustrate a method of bridging these two methodologies, by starting with a disulfide bridged phage display peptide which binds a human antibody Fc fragment (Delano et al. Science 2000, 287, 1279) and creating a backbone cyclic beta-hairpin peptidomimetic with 80-fold higher affinity for the Fc domain. The peptidomimetic is shown to adopt a well-defined beta-hairpin conformation in aqueous solution, with a bulge in one beta-strand, as seen in the crystal structure of the phage peptide bound to the Fc domain. The higher binding affinity of the peptidomimetic presumably reflects the effect of constraining the free ligand into the conformation required for binding, thus highlighting in this example the influence that ligand flexibility has on the binding energy. Since phage display peptides against a wide variety of different proteins are now accessible, this approach to synthetic ligand design might be applied to many other medicinally and biotechnologically interesting target proteins.     

Generation of anti-colorectal cancer fab phage display libraries with a high percentage of diverse antigen-reactive clones

A combinatorial Fab phage display library was generated from the antibody variable region genes of each of 2 BALB/c mice immunized with the human colorectal cancer cell lines SW480, SW948, and SW837. These libraries were shown to be diverse by nucleotide sequencing and diagnostic restriction enzyme digestion (fingerprinting) of individual members. The two libraries were combined and selected for binding to a suspension of formaldehyde-fixed human colorectal cancer cells in two successive rounds of selection and phage amplification by infection of bacteria. Analysis of the selected libraries as well as individual library clones by ELISA, showed binding to the cancer cell lines in both formaldehyde-fixed and native forms. Fifty five percent and 94% of library clones were positive for colorectal cancer cell binding after the first and second rounds of selection, respectively. Fingerprinting of individual clones showed the first round selected library to be very diverse and the second round selected library to be of more limited diversity. After absorption with normal human cell types, these anti-cancer selected libraries could be used to develop therapeutic and/or diagnostic agents.     

From phage display to dendrimer display: insights into multivalent binding

Phage display is widely used for the selection of target-specific peptide sequences. Presentation of phage peptides on a multivalent platform can be used to (partially) restore the binding affinity. Here, we present a detailed analysis of the effects of valency, linker choice, and receptor density on binding affinity of a multivalent architecture, using streptavidin (SA) as model multivalent receptor. For surfaces with low receptor densities, the SA binding affinity of multivalent dendritic phage peptide constructs increases over 2 orders of magnitude over the monovalent species (e.g., K(d,mono) = 120 μM vs K(d,tetra) = 1 μM), consistent with previous work. However, the affinity of the SA-binding phage presenting the exact same peptides was 16 pM when dense receptor surfaces used for initial phage display were used in assays. The phage affinity for SA-coated surfaces weakens severely toward the nanomolar regime when surface density of SA is decreased. A similarly strong dependence in this respect was observed for dendritic phage analogues. When presented with a dense SA-coated surface, dendrimer display affords up to a 10(4)-fold gain in affinity over the monovalent peptide. The interplay between ligand valency and receptor density is a fundamental aspect of multivalent targeting strategies in biological systems. The perspective offered here suggests that in vivo targeting schemes might best be served to conduct ligand selection under physiologically relevant receptor density surfaces, either by controlling the receptor density placed at the selection surface or by using more biologically relevant intact cells and tissues.     

Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity

BACKGROUND: Molecular diversity in nature is developed through a combination of genetic and chemical elements. We have developed a method that permits selective manipulation of both these elements in one protein engineering tool. It combines the ability to introduce non-natural amino acids into a protein using native chemical ligation with exhaustive targeted mutagenesis of the protein via phage-display mutagenesis. RESULTS: A fully functional biosynthetic version of the protease inhibitor eglin c was constructed. The amino-terminal fragment (residues 8-40) was chemically synthesized with a non-natural amino acid at position 25. The remaining carboxy-terminal fragment was expressed as a 30-residue peptide extension of gIIIp or gVIIIp on filamentous phage in a phage-display mutagenesis format. Native chemical ligation was used to couple the two fragments and produced a protein that refolded to its active form. To facilitate the packing of the introduced non-natural amino acid, residues 52 and 54 in the carboxy-terminal fragment were fully randomized by phage-display mutagenesis. Although the majority of the observed solutions for residues 52 and 54 were hydrophobic - complementing the stereochemistry of the introduced non-natural amino acid - a significant number of residues (unexpected because of stereochemical and charge criteria) were observed in these positions. CONCLUSIONS: Peptide synthesis and phage-display mutagenesis can be combined to produce a very powerful protein engineering tool. The physical properties of the environment surrounding the introduced non-natural residue can be selected for by evaluating all possible combinations of amino acid types at a targeted set of sites using phage-display mutagenesis.     

Identification of small molecule binding sites within proteins using phage display technology

Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.     

Potential of peptides selected from random phage-displayed libraries to mimic conformational epitopes: a study on scorpion toxin Cn2 and the neutralizing monoclonal antibody BCF2

Many conformational epitopes cannot be mapped by the use of a phage display approach due to the lack of amino acid similarity with the selected peptides. Exploring the potential of the method, we selected mimotopes of the discontinuous, highly conformational epitope of scorpion neurotoxin Cn2, whose 3D structure is known, using its generic neutralizing monoclonal antibody BCF2. With an exhaustive selection procedure, we isolated from a 12-mer phage library a large collection of mimotopes that reproduce the antigenic and immunogenic specificity of the Cn2-epitope. The selected peptides presented three sequence motifs, the most abundant of which, RD(N)XXGF, appeared in 15 different sequence contexts displayed by 97 out of 206 clones. In the most reactive mimotope, displayed by 24 (25%) clones, the motif was flanked by two Cys residues allowing the adoption of a cyclic conformation. Motifs QL(H,M)L(M) and (S/T)WHLP were selected with less efficiency. Comparison of the motifs with the primary and three-dimensional structure of Cn2 as well as with a model of the Cn2-BCF2(Fv) complex suggests that RD(N)XXGF, which does not share sequence similarity with the epitope, mimics its central structural element, turn 7-11, by using an alternative amino acid combination nevertheless keeping the nature of its interactions with BCF2. The QL(H,M)L(M) is assumed to mimic the hydrophobic part of the epitope. The principles of the conformational mimicry by phage-displayed peptides are discussed.     

Probing the conformational features of a phage display polypeptide sequence directed against single-walled carbon nanohorn surfaces

Single-walled carbon nanohorns (SWNHs) are interesting carbon nanostructures that have applications to science and technology. Using M13 phage display technology, polypeptides directed again SWNHs surfaces have been created for a number of nanotechnology and pharmaceutical purposes, yet the molecular mechanism of polypeptide sequence interaction and binding to SWNHs surfaces is not known. Recently, we identified a linear 12-AA M13 phage pIII sequence, NH-12-5-2 (DYFSSPYYEQLF), that binds with high affinity to SWNHs surfaces. To probe the structure of this pIII tail polypeptide further, we investigated the conformation of a model peptide representing the 12 AA NH-12-5-2 sequence. At neutral pH, the NH-12-5-2 model polypeptide is conformationally labile and exhibits two-state conformational exchange involving the D1-S5 N-terminal segment. Simultaneous with this conformational exchange process is the observation that the P6 residue exhibits imido ring conformational variation. In the presence of the structure-stabilizing solvent, TFE, or at pH 2.5, both the exchange process and Pro ring motion phenomena disappear, indicating that the structure of this peptide sequence can be stabilized by extrinsic factors. Interestingly, we observe NMR parameters (ROEs, (3)J coupling constants) for NH-12-5-2 in 90% v/v TFE that are consistent with the presence of a partial helical structure, similar to what was observed at low pH in our earlier CD experiments. We conclude that the NH-12-5-2 model polypeptide sequence possesses an inherent conformational instability that involves the D1-S5 sequence segment and the P6 residue but that this instability can be offset by extrinsic factors (e.g., charge neutralization, imido ring interconversion, and hydrophobic-hydrophobic interactions). These nonbonding interactions may play a role in the recognition and binding of this phage sequence region to SWNHs surfaces.     

Random sequence libraries displayed on phage: identification of biologically important molecules

Phage display has become a widely used tool for the identification of proteins or peptides with affinity for a variety of biomolecules. The versatility, simplicity and cost effectiveness of this application has pervaded a wide variety of research areas. Although not without its limitations, phage display has provided a convenient methodology for obtaining ligands to study the function, structure and diagnostic or therapeutic potential of various macromolecules. This review highlights some recent research employing this technology that serves to illustrate its utility in various research and clinical applications.     

MimoDB: a new repository for mimotope data derived from phage display technology

Peptides selected from phage-displayed random peptide libraries are valuable in two aspects. On one hand, these peptides are candidates for new diagnostics, therapeutics and vaccines. On the other hand, they can be used to predict the networks or sites of protein-protein interactions. MimoDB, a new repository for these peptides, was developed, in which 10,716 peptides collected from 571 publications were grouped into 1,229 sets. Besides peptide sequences, other important information, such as the target, template, library and complex structure, was also included. MimoDB can be browsed and searched through a user-friendly web interface. For computational biologists, MimoDB can be used to derive customized data sets and benchmarks, which are useful for new algorithm development and tool evaluation. For experimental biologists, their results can be searched against the MimoDB database to exclude possible target-unrelated peptides. The MimoDB database is freely accessible at http://immunet.cn/mimodb/.     

Selection of v-abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display

Methodologies for rapidly identifying cellular protein interactions resulting in posttranslational modification of one of the partners are lacking. Here, we select for substrates of the v-abl tyrosine kinase from two protein display libraries in which the protein is covalently linked to its encoding mRNA. Successive selection cycles from a randomized peptide library identified a consensus sequence closely matching that previously reported for the v-abl tyrosine kinase. Selections from a proteomic library derived from cellular mRNA identified several novel targets of v-abl, including a new member of a class of SH2 domain-containing adaptor proteins. Upon modification, several of the substrates obtained in these selections were found to be effective inhibitors of v-abl kinase activity in vitro. These experiments establish a novel method for identifying the substrates of tyrosine kinases from synthetic and cellular protein libraries.