Recombinant Peptide Libraries Research Articles

Design of Tetra-Peptide Ligands of Antibody Fc Regions Using In Silico Combinatorial Library Screening.

Peptides, or short chains of amino-acid residues, are becoming increasingly important as active ingredients of drugs and as crucial probes and/or tools in medical, biotechnological, and pharmaceutical research. Situated at the interface between small molecules and larger macromolecular systems, they pose a difficult challenge for computational methods. We report an in silico peptide library generation and prioritization workflow using CmDock for identifying tetrapeptide ligands that bind to Fc regions of antibodies that is analogous to known in vitro recombinant peptide libraries' display and expression systems. The results of our in silico study are in accordance with existing scientific literature on in vitro peptides that bind to antibody Fc regions. In addition, we postulate an evolving in silico library design workflow that will help circumvent the combinatorial problem of in vitro comprehensive peptide libraries by focusing on peptide subunits that exhibit favorable interaction profiles in initial in silico peptide generation and testing.

Evolving a Peptide: Library Platforms and Diversification Strategies.

Peptides are widely used in pharmaceutical industry as active pharmaceutical ingredients, versatile tools in drug discovery, and for drug delivery. They find themselves at the crossroads of small molecules and proteins, possessing favorable tissue penetration and the capability to engage into specific and high-affinity interactions with endogenous receptors. One of the commonly employed approaches in peptide discovery and design is to screen combinatorial libraries, comprising a myriad of peptide variants of either chemical or biological origin. In this review, we focus mainly on recombinant peptide libraries, discussing different platforms for their display or expression, and various diversification strategies for library design. We take a look at well-established technologies as well as new developments and future directions.

Multiplex localization of sequential peptide epitopes by use of a planar microbead chip

Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28γ (proteasome activator 28γ) polyclonal serum, for a murine monoclonal antibody against PA28γ, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks.

In vitro methods for peptide display and their applications

The presentation of recombinant peptide libraries linked to their coding sequence can be referred to as 'peptide display'. Phage display is the most widely practiced peptide display technology but more recent alternatives such as CIS display, ribosome display and mRNA display offer advantages over phage for speed, library size and the display of unnatural amino acids. These have provided researchers with tools to address some of the failings of peptides such as their low affinity, low stability and inability to cross biological membranes. In this review, we assess some of the recent advances in peptide display and its application.

Peptide mimicking antigenic and immunogenic epitope of neuwiedase from Bothrops neuwiedi snake venom

Peptides derived from a phage display library may mimic essential features of epitopes (mimotopes), including their immunogenicity. A recombinant peptide library of 12 amino acids displayed on the phage capsid was used to obtain peptides that mimic epitopes of antigens that are reactive to specific polyclonal antibodies anti-neuwiedase (NEU), a toxin from Bothrops neuwiedi snake venom. These polyclonal antibodies are protective against NEU activity and were used as target for the peptide library biopannings, resulting in the selection of 80 peptides. Antibody-binding epitopes were obtained by sequence alignment with the primary and tertiary structures of the NEU protein. Antigenicity and specificity of the mimotopes mixture were confirmed by dot blot, immuno dot blot, plaque reduction and Western blot assays. Their immunogenicity was demonstrated by immunization of BALB/c mice and ELISA tests. The NEU toxin is an important antigen that has many common structural regions to several toxic venom metalloproteinases, in which two epitope regions have been detected. The two mapped epitopes were found in primary sequences of several snake venom toxins, thus demonstrating the potential application of these NEU mimotopes as possible antigen components that are toxicity free.

Recombinant peptides targeted to the radiation-inducible response in tumor microvasculature

Recombinant peptides targeted to the radiation-inducible response in tumor microvasculature

Recombinant peptides targeted to the radiation-inducible response in tumor microvasculature

Recombinant peptides targeted to the radiation-inducible response in tumor microvasculature

Requirement for both H-2Db and H-2Kd for the induction of diabetes by the promiscuous CD8+ T cell clonotype AI4.

The NOD mouse is a model for autoimmune type 1 diabetes in humans. CD8(+) T cells are essential for the destruction of the insulin-producing pancreatic beta cells characterizing this disease. AI4 is a pathogenic CD8(+) T cell clone, isolated from the islets of a 5-wk-old female NOD mouse, which is capable of mediating overt diabetes in the absence of CD4(+) T cell help. Recent studies using MHC-congenic NOD mice revealed marked promiscuity of the AI4 TCR, as the selection of this clonotype can be influenced by multiple MHC molecules, including some class II variants. The present work was designed, in part, to determine whether similar promiscuity also characterizes the effector function of mature AI4 CTL. Using splenocyte and bone marrow disease transfer models and in vitro islet-killing assays, we report that efficient recognition and destruction of beta cells by AI4 requires the beta cells to simultaneously express both H-2D(b) and H-2K(d) class I MHC molecules. The ability of the AI4 TCR to interact with both H-2D(b) and H-2K(d) was confirmed using recombinant peptide libraries. This approach also allowed us to define a mimotope peptide recognized by AI4 in an H-2D(b)-restricted manner. Using ELISPOT and mimotope/H-2D(b) tetramer analyses, we demonstrate for the first time that AI4 represents a readily detectable T cell population in the islet infiltrates of prediabetic NOD mice. Our identification of a ligand for AI4-like T cells will facilitate further characterization and manipulation of this pathogenic and promiscuous T cell population.

Characterization and identification of allergen epitopes: recombinant peptide libraries and synthetic, overlapping peptides

For the understanding of the relationship between protein structure and allergenicity, it is important to identify allergenic epitopes. Two methods to characterize primarily linear epitopes are compared using the major allergen from brown shrimp ( Penaeus aztecus), Pen a 1, as an example. A recombinant peptide library was constructed and synthetic, overlapping peptides, spanning the entire Pen a 1 molecule, were synthesized and tested for specific IgE reactivity. Both methods identified IgE-binding of Pen a 1, however, the SPOTs procedure resulted in the identification of more epitopes of the major shrimp allergen Pen a 1 than the usage of the recombinant peptide library. For detection of specific IgE antibodies, the usage of 125I-labeled detection antibody seems to be superior over enzyme-labeled anti IgE antibodies. The regeneration of SPOTs membranes is possible, but it is prudent to test regenerated membranes for residual activity. If a given food allergen contains significant linear epitopes, which seems to be true for stable major allergens such as those of peanut and shrimp the SPOTs system may be more advantageous than the use of recombinant peptides libraries. However, if allergens are studied that contain more conformational epitopes, recombinant peptide libraries may help to identify the relevant epitopes. It has to be emphasized that no system for epitope identification will detect all epitopes and that the relevance of identified epitopes has to be confirmed with other methods such as inhibition studies, crystallographic analysis or the immunological evaluation of modified whole allergens.

Targeting growth factor and cytokine receptors with recombinant peptide libraries

The single-transmembrane-spanning receptors of cytokines and growth factors have historically proven resistant to the small-molecule screening efforts of the pharmaceutical industry. Advances in combinatorial library approaches to ligand discovery have begun to show success with these targets. There are several recent reports of peptides, derived from randomly assembled collections of L-peptides expressed in recombinant display vectors, that are high-affinity antagonists and even agonists of these receptors. These results indicate that molecules much smaller than the natural protein factors can interact effectively with these receptors, and this may lead the way to the discovery of even smaller nonpeptidic agents.

Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine.

Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.

High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries.

Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.

Peptides Which Bind to E-selectin and Block Neutrophil Adhesion

E-selectin is an inducible cell adhesion molecule which mediates rolling of neutrophils on the endothelium, an early event in the development of an inflammatory response. Inhibition of selectin-mediated rolling is a possible means for controlling inflammation-induced diseases, and several classes of compounds have been tested for this use. We describe here the use of recombinant peptide library screening for identification and optimization of novel ligands which bind to E-selectin. Several of these peptides bind with Kd values in the low nanomolar range and block E-selectin-mediated adhesion of neutrophils in static and flow-cell assays. Administration of the peptide to mice undergoing an acute inflammatory response reduced the extent of neutrophil transmigration to the site of inflammation, demonstrating the utility of this compound as a potential therapeutic. The identification of a peptide ligand for E-selectin suggests that the complete natural ligand for this adhesion molecule may include protein as well as carbohydrate moieties.