Identification of gliadin-binding peptides by phage display

Background  

Coeliac disease (CD) is a common and complex disorder of the small intestine caused by intolerance to wheat gluten and related edible cereals like barley and rye. Peptides originating from incomplete gliadin digestion activate the lamina propria infiltrating T cells to release proinflammatory cytokines, which in turn cause profound tissue remodelling of the small intestinal wall. There is no cure for CD except refraining from consuming gluten-containing products.     

Selection of silk-binding peptides by phage display

Peptides that bind to silkworm-derived silk fibroin fiber were selected from a phage-displayed random peptide library. The selected silk-binding peptides contained a consensus sequence QSWS which is important for silk-binding as confirmed by binding assays using phage and synthetic peptides. With further optimization, we anticipate that the silk-binding peptides will be useful for functionalization of silk for biomaterial applications.     

Lipopolysaccaride-binding peptides obtained by phage display method

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. It has strong toxicity and might cause sepsis or septic shock. Thus early detection of LPS and neutralization of LPS toxicity are required. We obtained several new LPS-binding peptides using a phage display method. We synthesized 3 of these peptides and analyzed their binding affinity and capacity to LPS. One of these peptides, named Li5-001, showed high binding affinity to LPS and lipid A; the K_d values were 10 and 1 nM, respectively. Li5-001 showed a high binding capacity to LPS, and was estimated to bind 130 ng LPS/mg, which is higher than that of polymyxin B (80 ng LPS/mg); however, its LPS-neutralizing activity was low. Li5-001 coupled with beads will be useful for eliminating endotoxin contamination from pharmaceuticals. Its low LPS-neutralizing activity allows to be used in the Limulus amebocyte lysate test without eluting LPS from the Li5-001 coupled beads.     

Selection of Burkholderia pseudomallei protease-binding peptides by phage display

Burkholderia pseudomallei is a causative agent of melioidosis, a fatal community acquired septicemia in Southeast Asia and Northern Australia. A protease has been proposed to be one of the major pathogenic factors to play a significant role in melioidosis. We have used phage display technology to identify peptides binding to B. pseudomallei protease. By screening a constrained cyclic heptapeptide library, five independent clones with affinity to this protease were isolated and the amino acid sequences were determined. The cyclic heptapeptides from two of the phage clones (Cys-Phe-Phe-Met-Pro-His-Thr-Phe-Cys) were identical and showed the strongest phage-protease interaction as detected by ELISA. Four of the five selected phages at the amount of 10¹³ phages could inhibit B. pseudomallei protease activity by approximately 50%.     

Construction and analysis of a genetically tuneable lytic phage display system

The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ.     

Characterization of prostate-specific antigen binding peptides selected by phage display technology

Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82-87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer.     

Aluminum- and mild steel-binding peptides from phage display

Using a phage library displaying random peptides of 12 amino acids on its surface, several peptides were found that bind to aluminum and mild steel. Like other metal-binding peptides, no obvious consensus motif has been found for these peptides. However, most of them are rich in hydroxyl-containing amino acids, serine or threonine, or contain histidine. For the aluminum-binding peptides, peptides with a higher number of hydroxyl-containing amino acids bind to the aluminum surface more tightly. For example, Val-Pro-Ser-Ser-Gly-Pro-Gln-Asp-Thr-Arg-Thr-Thr, which contains five hydroxyl-containing amino acid residues, was selected four-fold more frequently than a peptide containing only one serine, suggesting an important role for the hydroxyl-containing amino acids in the metal–peptide interaction.     

Amyloid-forming peptides selected proteolytically from phage display library

We demonstrated that amyloid-forming peptides could be selected from phage-displayed library via proteolysis-based selection protocol. The library of 28-residue peptides based on a sequence of the second zinc finger domain of Zif268, and computationally designed betabetaalpha peptide, FSD-1, was presented monovalently on the surface of M13 phage. The library coupled the infectivity of phage particles to proteolytic stability of a peptide introduced into the coat protein III linker. It was designed to include variants with a strong potential to fold into betabetaalpha motif of zinc finger domains, as expected from secondary structure propensities, but with no structure stabilization via zinc ion coordination. As our primary goal was to find novel monomeric betabetaalpha peptides, the library was selected for stable domains with the assumption that folded proteins are resistant to proteolysis. After less than four rounds of proteolytic selection with trypsin, chymotrypsin, or proteinase K, we obtained a number of proteolysis-resistant phage clones containing several potential sites for proteolytic attack with the proteinases. Eight peptides showing the highest proteolysis resistance were expressed and purified in a phage-free form. When characterized, the peptides possessed proteolytic resistance largely exceeding that of the second zinc finger domain of Zif268 and FSD-1. Six of the characterized peptides formed fibrils when solubilized at high concentrations. Three of them assembled into amyloids as determined through CD measurements, Congo red and thioflavin T binding, and transmission electron microscopy.     

Streptavidin-binding and -dimerizing ligands discovered by phage display, topochemistry, and structure-based design

Structural and mechanistic determinants of affinity of streptavidin-binding peptide ligands discovered by phage display are reviewed along with the use of streptavidin as a paradigm for structure-based design. A novel way of producing protein-dimerizing ligands in the streptavidin model system is discussed, in which crystal packing topochemically mediates or even catalyzes dimerization of adjacent bound ligands whose reactive ligating groups are presented toward one another in productive orientations in the crystal lattice. Finally, through crystallography on a set of streptavidin complexes with small molecule and peptide ligands at multiple pHs in two space groups, the mechanism by which ligands enhance intersubunit stabilization of the streptavidin tetramer is probed.     

Fibrin specific peptides derived by phage display: characterization of peptides and conjugates for imaging

Peptides that bind to fibrin but not to fibrinogen or serum albumin were selected from phage display libraries as targeting moieties for thrombus molecular imaging probes. Three classes of cyclic peptides (cyclized via disulfide bond between two Cys) were identified with consensus sequences XArXCPY(G/D)LCArIX (Ar = aromatic, Tn6), X(2)CXYYGTCLX (Tn7), and NHGCYNSYGVPYCDYS (Tn10). These peptides bound to fibrin at ～2 sites with K(d) = 4.1 μM, 4.0 μM, and 8.7 μM, respectively, whereas binding to fibrinogen was at least 100-fold weaker. The peptides also bind to the fibrin degradation product DD(E) with similar affinity to that measured for fibrin. The Tn7 and Tn10 peptides bind to the same site on fibrin, while the Tn6 peptides bind to a unique site. Alanine scanning identified the N- and C-terminal ends of the Tn6 and Tn7 peptides as most tolerant to modification. Peptide conjugates with either fluorescein or diethylenetriaminepentaaceto gadolinium(III) (GdDTPA) at the N-terminus were prepared for potential imaging applications, and these retained fibrin binding affinity and specificity in plasma. Relaxivity and binding studies on the GdDTPA derivatives revealed that an N-terminal glycyl linker had a modest effect on fibrin affinity but resulted in lower fibrin-bound relaxivity.     

以表位模拟肽为亲和靶标的肉毒毒素受体结合抑制剂的筛选及鉴定

以设计合成的A型肉毒毒素表位模拟肽为亲和靶标,对噬菌体随机肽库进行筛选,寻找能与A型肉毒毒素表位模拟肽特异结合并能拮抗毒素毒性效应的分子,通过ELISA鉴定阳性克隆,并对鉴定的阳性克隆进行特异性分析及DNA测序。氨基酸序列同源性分析发现,针对P4、P5表位模拟肽获得了两条特异的结合序列,并通过动物保护实验在噬菌体展示肽水平对特异结合分子的毒素毒性拮抗效应进行了初步研究,初步证明结合肽对A型肉毒毒素有一定的保护作用。     

Selection of biologically active peptides by phage display of random peptide libraries

Random peptide libraries displayed on phage are used as a source of peptides for epitope mapping, for the identification of critical amino acids responsible for protein—protein interactions and as leads for the discovery of new therapeutics. Efficient and simple procedures have been devised to select peptides binding to purified proteins, to monoclonal and polyclonal antibodies and to cell surfaces in vivo and in vitro.     

Combinatorial discovery of tumor targeting peptides using phage display

Peptides possess appropriate pharmacokinetic properties to serve as cancer imaging or therapeutic targeting agents. Currently, only a small number of rationally-derived, labeled peptide analogues that target only a limited subset of antigens are available. Thus, finding new cancer targeting peptides is a central goal in the field of molecular targeting. Novel tumor-avid peptides can be efficiently identified via affinity selections using complex random peptide libraries containing millions of peptides that are displayed on bacteriophage. In vitro and in situ affinity selections may be used to identify peptides with high affinity for the target antigen in vitro. Unfortunately, it has been found that peptides selected in vitro or in situ may not effectively target tumors in vivo due to poor peptide stability and other problems. To improve in vivo targeting, methodological combinatorial chemistry innovations allow selections to be conducted in the environment of the whole animal. Thus, new targeting peptides with optimal in vivo properties can be selected in vivo in tumor-bearing animals. In vivo selections have been proven successful in identifying peptides that target the vasculature of specific organs. In addition, in vivo selections have identified peptides that bind specifically to the surface of or are internalized into tumor cells. In the future, direct selection of peptides for cancer imaging may be expedited using genetically engineered bacteriophage libraries that encode peptides with intrinsic radiometal-chelation or fluorescent sequences.     

Identification of metal ion binding peptides containing unnatural amino acids by phage display

The bidentate metal binding amino acid bipyridylalanine (BpyAla) was incorporated into a disulfide linked cyclic peptide phage displayed library to identify metal ion binding peptides. Selection against Ni²⁺–nitrilotriacetic acid (NTA) enriched for sequences containing histidine and BpyAla. BpyAla predominated when selections were carried out at lower pH, consistent with the differential pK_a’s of histidine and BpyAla. Two peptides containing BpyAla were synthesized and found to bind Ni²⁺ with low micromolar dissociation constants. Incorporation of BpyAla and other metal binding amino acids into peptide and protein libraries should enable the evolution of novel binding and catalytic activities.     

An improved phage display procedure for identification of lipopolysaccharide-binding peptides

We successfully implemented several modifications to the regular phage display procedure and significantly improved the lipopolysaccharides-binding properties of the peptides selected. Specifically, the number of biopannings was increased and peptides with consensus sequences were obtained. A dual selection procedure (referred to as subtractive panning) was used to simultaneously select for the desired target and deselect for an undesired target, thereby increasing the binding specificity. In addition, binding and washing conditions in the subtractive panning were also modified to favor the selection of peptides with higher binding strength. As a result, two peptides, ASFPPAF and SSHTISF, were identified with much improved binding properties compared to those selected with regular panning. The binding specificities of these two peptides, as measured by the ratio of phages bound to the desired and undesired targets, were severalfold higher than previously reported. These modifications could easily be implemented with many other target molecules, indicating the general applicability of the procedure.     

Functional cloning by phage display

This review focusses on the isolation of proteins from genomic or cDNA expression products libraries displayed on phage. The use of phage display is highlighted for the characterization of binding proteins with diverse biological functions. Phage display is compared with another strategy, the yeast two-hybrid method. The combination of both strategies is especially powerful to eliminate false positives and to get information on the biochemical functions of proteins.     

Affinity adsorbent based on combinatorial phage display peptides that bind alpha-cobratoxin

Combinatorial phage display was used to discover peptides that selectively bind to the alpha-cobratoxin (neurotoxin) component of the multi-component venom of the Thai cobra, Naja kaouthia. Peptide sequences determined in this way were synthesized chemically and were covalently attached to agarose through the alpha-amino terminus. Such affinity chromatography supports selectively bound the alpha-cobratoxin component from crude venom, while passage of the crude venom over the support selectively depleted the venom of this component. The selective binding of alpha-cobratoxin to peptide-based solid-phase supports suggests that a limitless variety of peptides similarly obtained by combinatorial phage display can be used to craft specific analytical and preparative tools.     

Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.     

Quantifying beta-sheet stability by phage display

Antigens I/II are large multifunctional adhesins from oral viridans streptococci that exert immunomodulatory effects on human cells and play important roles in inflammatory disorders. Among them, Streptococcus mutans plays a major role in the initiation of dental caries. The structure of the V-region (SrV+, residues 464-840) of the antigen I/II of S. mutans has been determined using the multiwavelength anomalous diffraction phasing technique with seleno-methionine-substituted recombinant protein and subsequently refined at 2.4 A resolution. The crystal structure of SrV+ revealed a lectin-like fold that displays a putative preformed carbohydrate-binding site stabilized by a metal ion. Inhibition of this binding site may confer to humans a protection against dental caries and dissemination of the bacteria to extra-oral sites involved in life-threatening inflammatory diseases. This crystal structure constitutes a first step in understanding the structure-function relationship of antigens I/II and may help in delineating new preventive or therapeutic strategies against colonization of the host by oral streptococci.