Screening phage-displayed combinatorial peptide libraries

Among the many techniques available to investigators interested in mapping protein-protein interactions is phage display. With a modest amount of effort, time, and cost, one can select peptide ligands to a wide array of targets from phage-display combinatorial peptide libraries. In this article, protocols and examples are provided to guide scientists who wish to identify peptide ligands to their favorite proteins.     

T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.     

Identification of calmodulin-binding peptide consensus sequences from a phage-displayed random peptide library

The calcium-binding protein, calmodulin (CaM), was used to screen a phage library displaying random peptides 26 amino acids (aa) in length. Twenty CaM-binding peptides were identified, 17 of which contained one of three consensus sequence motifs: + W-OλR, WRAAV or WRXXAAAL, where +, -, O,λ and X are positively charged, negatively charged, hydrophobic, leucine or valine, and any residue, respectively. The Trp residue in these motifs is located within 14 aa of the N-terminus of the displayed peptide. Previous studies [Dedman et al., J. Biol. Chem. 268 (1993) 23025–23030] using a library displaying random peptides 15 aa in length identified CaM-binding peptides which contained a Trp-Pro dipeptide motif. These results suggest that the type of CaM-binding motif identified can vary between different types of combinatorial peptides     

Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries

Signaling complexes usually involve multidomain proteins containing catalytic domains and peptide recognition modules (PRMs), which mediate protein-protein interactions and assemble complexes by binding to ligands containing a core sequence motif. Concomitant to large-scale physical interaction screening, considerable effort has been devoted toward the elucidation of consensus profiles for common PRMs. We describe herein a robust and proven protocol to generate consensus profiles for PRMs using phage-displayed peptide libraries. The initial phase of the protocol entails the cloning, expression and purification of PRMs as fusion proteins, in addition to the construction of highly diverse phage-displayed peptide libraries. The affinity selection process described thereafter enables a single researcher to efficiently probe the recognition profiles of numerous PRMs in a 1 week time period.     

Specific and selective probes for Pseudomonas aeruginosa from phage-displayed random peptide libraries

The design of novel biosensors for the detection of biological threats, such as Pseudomonas aeruginosa, requires probes that specifically bind biological agents and insure their immediate and efficient recognition. Advanced bio-selective sensors may meet the requests for isolation, concentration of the agents and their real-time detection. There is a need for robust and inexpensive affinity probes alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we identified from two phage-displayed random peptide libraries phage clones displaying peptides capable of specific and strong binding to P. aeruginosa cell surface. The ability of the phage clones to interact specifically with P. aeruginosa was demonstrated by using enzyme-linked immunosorbent assay (ELISA). We assessed selectivity of phage-bacteria-binding by comparing the binding ability of the selected clones to the selector bacterium and a panel of other bacterial species; we also demonstrated by dot spot and immunoblotting that the most reactive and selective phage peptide bound with high avidity the bacterial cell surface. In addition, as proof-of-concept, we tested the possibility to immobilize the affinity-selected phage to a putative biosensor surface. The quality of phage deposition was monitored by ELISA, and phage-bacterial-binding was confirmed by high-power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including clinical-based diagnostics and possibly biological warfare applications.     

Selecting peptides for use in nanoscale materials using phage-displayed combinatorial peptide libraries

Materials that combine inorganic components and biological molecules provide a new paradigm for synthesizing nanoscale and larger structures with tailored physical properties. These synthesis techniques utilize the molecular recognition properties of many biological molecules to nucleate and control growth of the nanoscale structure. Phage-displayed peptide libraries are a powerful tool to identify peptides that selectively recognize and bind to a variety of inorganic surfaces that are utilized in electronic and photonic devices. These libraries have been used extensively to study the peptide-mediated nucleation and growth of some metallic and semiconducting materials, and the application to designed nanostructures has been demonstrated.     

Selection of peptidic mimics of digoxin from phage-displayed peptide libraries by anti-digoxin antibodies

Since the initial report of the development of methodology to generate high-affinity digitalis-specific (digoxin) antibodies, these antibodies have proven extremely useful tools to monitor digoxin levels in digitalized patients and, as Fab fragments, to reverse toxic digoxin effects in life-threatening digoxin overdoses. These antibodies (both digoxin-specific and ouabain-specific) have been used extensively by investigators for the identification and characterization of putative endogenous digitalis-like factors. In this study, we used two well-characterized mouse anti-digoxin monoclonal antibodies (mAbs), designated 26-10 and 45-20, as binding templates with which to select short bacteriophage-displayed (pIII protein inserted) peptides that are capable of binding to these mAbs and mimicking the conformational structure of digoxin. Selective enrichment from two phage-displayed random peptide libraries enabled us to isolate and identify distinct 15 and 26 amino acid residue peptide inserts that bind with high avidity and idiotypic specificity to the selecting mAbs. Among these displayed inserts a subset was identified whose mAb binding is inhibited by digoxin and whose corresponding synthetic peptides inhibit phage binding. They, therefore, appear to bind at the mAbs digoxin-binding sites. These data provide the first clear evidence that short polypeptides can serve as surrogates for the low molecular mass hapten digoxin.     

New inhibitors of Helicobacter pylori urease holoenzyme selected from phage-displayed peptide libraries.

Urease is an important virulence factor for Helicobacter pylori and is critical for bacterial colonization of the human gastric mucosa. Specific inhibition of urease activity has been proposed as a possible strategy to fight this bacteria which infects billions of individual throughout the world and can lead to severe pathological conditions in a limited number of cases. We have selected peptides which specifically bind and inhibit H. pylori urease from libraries of random peptides displayed on filamentous phage in the context of pIII coat protein. Screening of a highly diverse 25-mer combinatorial library and two newly constructed random 6-mer peptide libraries on solid phase H. pylori urease holoenzyme allowed the identification of two peptides, 24-mer TFLPQPRCSALLRYLSEDGVIVPS and 6-mer YDFYWW that can bind and inhibit the activity of urease purified from H. pylori. These two peptides were chemically synthesized and their inhibition constants (Ki) were found to be 47 microM for the 24-mer and 30 microM for the 6-mer peptide. Both peptides specifically inhibited the activity of H. pylori urease but not that of Bacillus pasteurii.     

Antagonists of Hsp16.3, a low-molecular-weight mycobacterial chaperone and virulence factor, derived from phage-displayed peptide libraries

The persistence of Mycobacterium tuberculosis is a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.     

Screening of phage-displayed peptide libraries with a monoclonal antibody raised against the F protein of the bovine respiratory syncytial virus

Summary Most of the monoclonal antibodies (MAbs) raised against the fusion (F) protein of the bovine respiratory syncytial virus recognize discontinuous epitopes on the protein. In order to find mimotopes of these epitopes, phage-displayed peptide libraries were screened with MAbs. The results obtained with MAb AL11C2 are described here. After four or five pannings, colony immunoscreening with AL11C2 allowed the isolation of positive clones that are specific for this monoclonal antibody. Four different sequences were determined on isolated phages, three of which are cysteine-constrained peptides in fusion with PVIII and one is a hexapeptide in fusion with PIII. In the case of the peptides containing two cysteines, the binding to AL11C2 was shown to be dependent on the presence of a disulfide bridge. The recombinant phages were also shown to inhibit the binding of AL11C2 to its natural antigen in a competitive ELISA assay.     

High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold

Phage-displayed synthetic antibody libraries were built on a single human framework by introducing synthetic diversity at solvent-exposed positions within the heavy chain complementarity-determining regions (CDRs). The design strategy of mimicking natural diversity using tailored codons had been validated previously with scFv libraries, which produced antibodies that bound to antigen, murine vascular endothelial growth factor (mVEGF), with affinities in the 100nM range. To improve library performance, we constructed monovalent and bivalent antigen-binding fragment (Fab) libraries, and explored different CDR-H3 diversities by varying the amino acid composition and CDR length. A Fab with sub-nanomolar affinity for mVEGF was obtained from a library with CDR-H3 diversity designed to contain all 20 naturally occurring amino acids. We then expanded the library by increasing the variability of CDR-H3 length and using tailored codons that mimicked the amino acid composition of natural CDR-H3 sequences. The library was tested against a panel of 13 protein antigens and high-affinity Fabs were obtained for most antigens. Furthermore, the heavy chain of an anti-mVEGF clone was recombined with a library of light chain CDRs, and the affinity was improved from low nanomolar to low picomolar. The results demonstrated that high-affinity human antibodies can be generated from libraries with completely synthetic CDRs displayed on a single scaffold.     

噬菌体展示肽在血管导向治疗方面的应用

介绍了一种利用噬菌体肽库的新技术－体内噬菌体展示（n vivo phage display)。这项技术是在活的动物体内进行的肽库筛选。将肽库通过静脉注射到动物体内,因为血管分子内皮的异质性,噬菌体可以选择性地导向不同组织,这样就可以筛到与特定组织特异结合的噬菌体展示肽。动物实验表明,前凋亡小肽和细胞毒素与导各肽偶联后 治疗效果。这项技术应用于临床,一定有助于肿瘤等疾病的导向治疗和造影技术的发展。     

Peptide vaccines and peptide libraries

Synthetic immunogens, containing built-in adjuvanticity, B cell, T helper cell and CTL epitopes or mimotopes, are ideal and invaluable tools to study the immune response with respect to antigen processing and presentation. This serves as a basis for the development of complete and minimal vaccines which do not need large carrier proteins, further adjuvants, liposome formulations or other delivery systems. Combinatorial peptide libraries, either completely random or characterized by one or several defined positions, are useful tools for the identification of the critical features of B cell epitopes and of MHC class I and class II binding natural and synthetic epitopes. The complete activity pattern of an O/Xn library with hundreds of peptide collections, each made up from billions of different peptides, represents the ranking of amino acid residues mediating contact to the target proteins of the immune system. Combinatorial libraries support the design of peptides applicable in vaccination against infectious agents as well as therapeutic tumour vaccines. Using the principle of lipopeptide vaccines, strong humoral and cellular immune responses could be elicited. The lipopeptide vaccines are heat-stable, non-toxic, fully biodegradable and can be prepared on the basis of minimized epitopes by modern methods of multiple peptide synthesis. The lipopeptides activate the antigen-presenting macrophages and B cells and have been recently shown to stimulate innate immunity by specific interaction with receptors of the Toll family.     

T-cell epitope prediction with combinatorial peptide libraries.

T cell receptors (TCR) recognize antigenic peptides in complex with the major histocompatibility complex (MHC) molecules and this trimolecular interaction initiates antigen-specific signaling pathways in the responding T lymphocytes. For the study of autoimmune diseases and vaccine development, it is important to identify peptides (epitopes) that can stimulate a given TCR. The use of combinatorial peptide libraries has recently been introduced as a powerful tool for this purpose. A combinatorial library of n-mer peptides is a set of complex mixtures each characterized by one position fixed to be a specified amino acid and all other positions randomized. A given TCR can be fingerprinted by screening a variety of combinatorial libraries using a proliferation assay. Here, we present statistical models for elucidating the recognition profile of a TCR using combinatorial library proliferation assay data and known MHC binding data.     

Development of an enzyme-linked immunosorbent assay for thiacloprid in soil and agro-products with phage-displayed peptide

A monoclonal antibody (3A5) that can recognize thiacloprid was produced, and a linear 8-residue peptide phage library was constructed. Six phage-displayed peptides were isolated from the linear 8-residue peptide phage library and a cyclic 8-residue peptide phage library. A phage enzyme-linked immunosorbent assay (ELISA) was developed to detect thiacloprid using a phage-displayed peptide. Under the optimal conditions, the half-maximal inhibition concentration (IC₅₀) and the limit of detection (IC₁₀) of the developed phage ELISA were 8.3 and 0.7 μg/L, respectively. Compared with the conventional ELISA, the sensitivity was improved more than 3-fold. The cross-reactivity (CR) was less than 0.08% for the tested structural analogues and was regarded as negligible. The recoveries of thiacloprid ranged from 80.3% to 116.3% in environmental and agricultural samples, which conformed to the requirements for residue detection. The amount of thiacloprid detected by phage ELISA in the samples was significantly correlated with that detected by high-performance liquid chromatography. The current study indicates that isolating phage-displayed peptides from phage display libraries is an alternative method for the development of a sensitive immunoassay and that the developed assay is a potentially useful tool for detecting thiacloprid in environmental and agricultural samples.     

Screening and identification of a specific peptide binding to cervical cancer cells from a phage-displayed peptide library

Objectives

To screen and identify the probe markers specifically binding to human cervical cancer, a phage-displayed 12-mer peptide library was used for biopanning of SiHa cells.

Results

After four rounds of whole-cell subtraction biopanning, the phage recovery was 21-fold higher (from 3.9 × 10^?5 to 8.3 × 10^?4) than that of the first round, and specific phage clones were significantly enriched. 57 randomly selected phage clones were tested by ELISA, and 36 phage clones were identified as positive clones. After sequencing of positive clones, six different peptide sequences were obtained and CSP3 showed best affinity and specificity to SiHa cells via immunofluorescence assay.

Conclusions

Peptide, CSP3, bound to SiHa cells specifically and sensitively. It may be a potential candidate for molecular imaging detection and targeting therapy of cervical cancer.

     

Selection and identification of a specific peptide binding to ovarian cancer cells from a phage-displayed peptide library

Biotechnology Letters - Ovarian cancer is one of the most fatal gynecological malignancies. It is emergently needed to select a novel molecular fragment as a targeting element for the future...