Peptide and peptidomimetic libraries

Various techniques for generation of peptide and peptidomimetic libraries are summarized in this article. Multipin, tea bag, and split-couple-mix techniques represent the major methods used to make peptides and peptidomimetics libraries. The synthesis of these libraries were made in either discrete or mixture format. Peptides and peptidomimetics combinatorial libraries were screened to discover leads against a variety of targets. These targets, including bacteria, fungus, virus, receptors, and enzymes were used in the screening of the libraries. Discovered leads can be further optimized by combinatorial approaches.     

Mimotopes of tumor-associated T-cell epitopes for cancer vaccines determined with combinatorial peptide libraries

Cytotoxic T-cells are the most important effector cells in immune responses against tumors. The identification of tumor-associated epitopes for these cells, therefore, has become a key aspect of the development of cancer vaccines. Here, we describe a new approach to the determination of tumor-associated T-cell epitopes which employs combinatorial peptide libraries with singly defined sequence positions in a randomized context. The analysis of the responses of a T-cell clone to these libraries yields the amino acid constituents of the epitope which can be combined to obtain mimotopes that are suitable as vaccine antigens for the induction of tumor-specific responses.     

Peptide crosslinked micelles: a new strategy for the design and synthesis of peptide vaccines

This report presents a new and simple methodology for the synthesis of multicomponent peptide vaccines, named the peptide crosslinked micelles (PCMs). The PCMs are core shell micelles designed to deliver peptide antigens and immunostimulatory DNA to antigen-presenting cells (APCs). They are composed of immunostimulatory DNA, peptide antigen, and a thiopyridal derived poly(ethylene glycol)-polylysine block copolymer. The peptide antigen acts as a crosslinker in the PCM strategy, which allows the peptide antigen to be efficiently encapsulated into the PCMs and also stabilizes them against degradation by serum components. Cell culture studies demonstrated that the PCMs greatly enhance the uptake of peptide antigens into human dendritic cells.     

Synthetic peptide vaccines

The review considers the stages of the development of synthetic peptide vaccines against infectious agents, novel approaches and technologies employed in this process, including bioinformatics, genomics, proteomics, large-scale peptide synthesis, high-throughput screening methods, the use of transgenic animals for modeling of human infections. An important role for the development and selection of efficient adjuvants for peptide immunogens is noted. The review contains examples of the developments of synthetic peptide vaccines against three infectious diseases (malaria, hepatitis C, and foot-and-mouth disease).     

Random-peptide libraries and antigen-fragment libraries for epitope mapping and the development of vaccines and diagnostics

Random peptide libraries and antigen-fragment libraries (also known as gene-fragment libraries) have been used to identify epitopes on protein antigens. These technologies promise to make significant contributions to diagnostic and vaccine development. Researchers in a number of labs have shown that phage selected from libraries with protective antibodies, raised against whole antigen, can be used as immunogens to stimulate antibody responses that bind native antigen and provide protection in vivo. Others have used the sera of patients with idiopathic diseases to screen libraries, and by this approach have identified candidate antigens involved in immune disease. These may prove useful for diagnosis and, possibly, in determining disease etiology.     

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.     

Peptide vaccines: fantasy or reality?

World Journal of Microbiology and Biotechnology -     

肽疫苗的研究进展

肽疫苗可分为3大类,即抗病毒相关肽疫苗、抗肿瘤相关肽疫苗、抗细菌及寄生虫感染的肽疫苗。肽疫苗具有安全、廉价、特异性强、易于保存和应用等特点。本主要介绍有关肽疫苗的设计、提高免疫原性及体内的稳定性、复合肽疫苗、临床评价等方面的研究和应用进展。     

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 libraries: at the crossroads of proteomics and bioinformatics

Peptide libraries offer a valuable means for providing functional information regarding protein-modifying enzymes and protein interaction domains. Library approaches have become increasingly useful as high-throughput strategies for the analysis of large numbers of new proteins identified as a result of genome-sequencing efforts. Recent developments in the field have produced faster methods with broadened applicability. Crucially, new computational and biochemical tools have emerged that facilitate identification of interaction partners and substrates for proteins on the basis of their peptide selectivity profiles. Such combinations of proteomics-scale experimental approaches with bioinformatics tools hold great promise for the elucidation of protein interaction networks and signal transduction pathways in living cells.     

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.     

How can peptide vaccines work?

Peptide antigens frequently induce antibodies which recognise the denatured form of a protein from which their sequences are derived. However, the ability to induce antibodies which crossreact with the native, fully folded form of the protein is less commonly observed. Although there is a growing number of examples in which this is the case, the ability to predict peptides having this property is extremely limited. Given the large surface areas involved in antibody/antigen interaction it is surprising that peptides could ever induce antibodies which would recognise the native protein well enough to have biological activity, such as the neutralization of infectivity. A mechanism is proposed to explain such observations which is compatible with many of the properties of antipeptide antibodies.     

Synthetic antibody libraries focused towards peptide ligands

Synthetic antibody libraries have proven immensely useful for the de novo isolation of antibodies without the need for animal immunization. Recently, focused libraries designed to recognize particular classes of ligands, such as haptens or proteins, have been employed to facilitate the selection of high-affinity antibodies. Focused libraries are built using V regions encoding combinations of canonical structures that resemble the structural features of antibodies that bind the desired class of ligands and sequence diversity is introduced at residues typically involved in recognition. Here we describe the generation and experimental validation of two different single-chain antibody variable fragment libraries that efficiently generate binders to peptides, a class of molecules that has proven to be a difficult target for antibody generation. First, a human anti-peptide library was constructed by diversifying a scaffold: the human variable heavy chain (V_H) germ line gene 3-23, which was fused to a variant of the human variable light chain (V_L) germ line gene A27, in which L1 was modified to encode the canonical structure found in anti-peptide antibodies. The sequence diversity was introduced into 3-23 (V_H) only, targeting for diversification residues commonly found in contact with protein and peptide antigens. Second, a murine library was generated using the antibody 26-10, which was initially isolated based on its affinity to the hapten digoxin, but also binds peptides and exhibits a canonical structure pattern typical of anti-peptide antibodies. Diversity was introduced in the V_H only using the profile of amino acids found at positions that frequently contact peptide antigens. Both libraries yielded binders to two model peptides, angiotensin and neuropeptide Y, following screening by solution phage panning. The mouse library yielded antibodies with affinities below 20 nM to both targets, although only the V_H had been subjected to diversification.     

Discovering peptide ligands using epitope libraries.

Epitope libraries are large collections of peptides. Each peptide is displayed on the surface of a bacteriophage particle and is encoded by a randomly mutated region of the phage genome, thus associating each unique peptide with the DNA molecule encoding it. Antibodies and other binding proteins are used to select specifically for rare, phage-bearing peptide ligands; sequencing of the corresponding viral DNA will reveal their amino acid sequences. Relatively high-affinity peptides for a variety of peptide- and non-peptide-binding ligates have been affinity-isolated from epitope libraries. This technology has been used to map epitopes on proteins and to find peptide mimics for non-peptide-binding ligates. The current challenge lies in developing epitope library technology so that tight-binding peptide ligands can be detected for a wider variety of ligates, including those that recognize folded proteins. Should this be accomplished, many powerful applications can be envisioned in the areas of drug design and the development of diagnostic markers and vaccines.     

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.     

Construction of high-complexity combinatorial phage display peptide libraries

Random peptide libraries displayed on the surface of filamentous bacteriophage are widely used as tools for the discovery of ligands for biologically relevant macromolecules, including antibodies, enzymes, and cell surface receptors. Phage display results in linkage of an affinity-selectable function (the displayed peptide) to the DNA encoding that function, allowing selection of individual binding clones by iterative cycles of in vitro panning and in vivo amplification. Critical to the success of a panning experiment is the complexity of the library: the greater the diversity of clones within the library, the more likely the library contains sequences that will bind a given target with useful affinity. A method for construction of high-complexity (> or = 10(9) independent clones) random peptide libraries is presented. The key steps are highly efficient binary ligation under conditions where the vector is relatively dilute, with only a modest molar excess of insert, followed by efficient electrotransformation into Escherichia coli. Library design strategies and a protocol for rapid sequence characterization are also presented.