An ephrin mimetic peptide that selectively targets the EphA2 receptor

Eph receptor tyrosine kinases represent promising disease targets because they are differentially expressed in pathologic versus normal tissues. The EphA2 receptor is up-regulated in transformed cells and tumor vasculature where it likely contributes to cancer pathogenesis. To exploit EphA2 as a therapeutic target, we used phage display to identify two related peptides that bind selectively to EphA2 with high affinity (submicromolar K(D) values). The peptides target the ligand-binding domain of EphA2 and compete with ephrin ligands for binding. Remarkably, one of the peptides has ephrin-like activity in that it stimulates EphA2 tyrosine phosphorylation and signaling. Furthermore, this peptide can deliver phage particles to endothelial and tumor cells expressing EphA2. In contrast, peptides corresponding to receptor-interacting portions of ephrin ligands bind weakly and promiscuously to many Eph receptors. Bioactive ephrin mimetic peptides could be used to selectively deliver agents to Eph receptor-expressing tissues and modify Eph signaling in therapies for cancer, pathological angiogenesis, and nerve regeneration.     

Consensus sequence L/PKSSLL mimics crucial epitope on Loop III of Taiwan cobra cardiotoxin

Phage display is effective in screening peptides that mimic venom’s neutralizing epitopes. A phage display cyclized heptapeptide library (C7C library) was panned with purified divalent antivenin IgG, which neutralizes Naja naja atra venom (NAV) and Bungarus multicinctus venom (BMV). The selected heptapeptide sequences were aligned with known protein sequences of NAV and BMV in GenBank. One of the four consensus sequences, L/PKSSLL, mimicked the crucial epitope on Loop III of Taiwan cobra cardiotoxin that is associated with the venom’s lethal potency. In dot blot analysis, several clones showed varying reactivities for NAV monovalent antivenin and lesser cross-reactions with BMV monovalent antivenin. The KSSLLRN-carrying phage occurred four times in selected clones and showed the strongest reactivity to NAV monovalent antivenin. Furthermore, the QDSLLPS-carrying phage also presented significant dot blot signal, indicating that the SLL sequence shared by these two clones may be a crucial antibody-binding site.     

Use of bacteriophage T7 displayed peptides for determination of monoclonal antibody specificity and biosensor analysis of the binding reaction

A heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of the monoclonal antibodies F4, F5, and LT1 directed against mouse polyomavirus large T-antigen. Phage selected by biopanning was cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay. In phage reacting with the F5 antibody the deduced amino acid sequence of the displayed peptides corresponded to a segment of large T-antigen. In phage reacting with the antibodies F4 and LT1, no such similarity was observed. The kinetics of phage particle-monoclonal antibody complex formation and dissociation was analyzed in an optical biosensor instrument. Sensor chips of standard quality were useful for binding analysis of T7 phage in crude lysates of infected Escherichia coli. We synthesized peptides corresponding to selected consensus sequences and showed by biosensor analysis that these peptides (linear NH3-CPNSLTPADPTMDY-COOH and NH3-NSLTPCNNKPSNRC-COOH with an intramolecular S--S bridge) were able to compete with large T-antigen in binding to the corresponding antibodies (LT1 and F4). These synthetic peptides were also used for gentle and specific dissociation of large T-antigen-antibody complexes. The results demonstrate the potential of phage T7 for display of peptides and for rapid analysis of interactions of these peptides with ligands.     

Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressors.

The filamentous phage coat protein pIII has been used to display a variety of peptides and proteins to allow easy screening for desirable binding properties. We have examined the biological constraints that restrict the expression of short peptides located in the early mature region of pIII, adjacent to the signal sequence cleavage site. Many functionally defective pIII fusion proteins contained several positively charged amino acids in this region. These residues appear to inhibit proper insertion of pIII into the Escherichia coli inner membrane, blocking the assembly and extrusion of phage particles. Suppressor mutations in the prlA (secY) component of the protein export apparatus dramatically alleviate the phage growth defect caused by the positively charged residues. We conclude that insertion of pIII fusion proteins into the inner membrane can occur by a sec gene-dependent mechanism. The suppressor strains should be useful for increasing the diversity of peptides displayed on pIII in phage libraries.     

In vitro system for high-throughput screening of random peptide libraries for antimicrobial peptides that recognize bacterial membranes.

Antibacterial peptides have been isolated from a wide range of species. Some of these peptides act on microbial membranes, disrupting their barrier function. With the increasing development of antibiotic resistance by bacteria, these antibacterial peptides, which have a new mode of action, have attracted interest as antibacterial agents. To date, however, few effective high-throughput approaches have been developed for designing and screening peptides that act selectively on microbial membranes. In vitro display techniques are powerful tools to select biologically functional peptides from peptide libraries. Here, we used the ribosome display system to form peptide-ribosome-mRNA complexes in vitro from nucleotides encoding a peptide library, as well as immobilized model membranes, to select specific sequences that recognize bacterial membranes. This combination of ribosome display and immobilized model membranes was effective as an in vitro high-throughput screening system and enabled us to identify motif sequences (ALR, KVL) that selectively recognized the bacterial membrane. Owing to host toxicity, it was not possible to enrich any sequence expected to show antimicrobial activity using another in vitro system, e.g. phage display. The synthetic peptides designed from these enriched motifs acted selectively on the bacterial model membrane and showed antibacterial activity. Moreover, the motif sequence conferred selectivity onto native peptides lacking selectivity, and decreased mammalian cell toxicity of native peptides without decreasing their antibacterial activity.     

Selection of internalizing ligand-display phage using rolling circle amplification for phage recovery

Selection of phage libraries against complex living targets such as whole cells or organs can yield valuable targeting ligands without prior knowledge of the targeted receptor. Our previous studies have shown that noninfective multivalent ligand display phagemids internalize into mammalian cells more efficiently than their monovalent counterparts suggesting that cell-based selection of internalizing ligands might be improved using multivalently displayed peptides, antibodies or cDNAs. However, alternative methods of phage recovery are needed to select phage from noninfective libraries. To this end, we reasoned that rolling circle amplification (RCA) of phage DNA could be used to recover noninfective phage. In feasibility studies, we obtained up to 1.5 million-fold enrichment of internalizing EGF-targeted phage using RCA. When RCA was applied to a large random peptide library, eight distinct human prostate carcinoma cell-internalizing peptides were isolated within three selection rounds. These data establish RCA as an alternative to infection for phage recovery that can be used to identify peptides from noninfective phage display libraries or infective libraries under conditions where there is the potential for loss of phage infectivity.     

Use of in vivo phage display to engineer novel adenoviruses for targeted delivery to the cardiac vasculature

We performed in vivo phage display in the stroke prone spontaneously hypertensive rat, a cardiovascular disease model, and the normotensive Wistar Kyoto rat to identify cardiac targeting peptides, and then assessed each in the context of viral gene delivery. We identified both common and strain-selective peptides, potentially indicating ubiquitous markers and those found selectively in dysfunctional microvasculature of the heart. We show the utility of the peptide, DDTRHWG, for targeted gene delivery in human cells and rats in vivo when cloned into the fiber protein of subgroup D adenovirus 19p. This study therefore identifies cardiac targeting peptides by in vivo phage display and the potential of a candidate peptide for vector targeting strategies.     

Screening,tandem expression and immune activity appraisal of <Emphasis Type="Italic">Deinagkistrodon acutus</Emphasis> (pit viper) venom mimotopes from a phage display 12-mer peptide library

Objective

To design a specific polyclonal antibody against Deinagkistrodon acutus venom (DA-pAb) by immunizating New Zealand white rabbits.

Results

The IgG fraction was purified by affinity chromatography, and specific antibodies were purified by immunoaffinity chromatography. Polyclonal antibodies were subjected to ELISA and western blotting to evaluate their immune reactivity. We identified the mimotopes by screening a phage display 12-mer peptide library against D. acutus venom. After three rounds of biopanning with DA-pAb, 30 positive clones were identified. Eighteen phage clones were sequenced, and their corresponding amino acid sequences were deduced. Additional immunoassays with the peptides and DA-pAb identified five sequences as possible epitopes. Recombinant antigens synthesized with the five epitopes were used for the immunization of BALB/c mice.

Conclusion

The antibodies induced by these peptides recognized the recombinant antigen and D. acutus venom and protected mice against the hemorrhagic effects of the venom.

     

Phage display can select over-hydrophobic sequences that may impair prediction of natural domain-peptide interactions

MOTIVATION: The phage display peptide selection approach is widely used for defining binding specificities of globular domains. PDZ domains recognize partner proteins via C-terminal motifs and are often used as a model for interaction predictions. Here, we investigated to which extent phage display data that were recently published for 54 human PDZ domains can be applied to the prediction of human PDZ-peptide interactions. RESULTS: Promising predictions were obtained for one-third of the 54 PDZ domains. For the other two-thirds, we detected in the phage display peptides an important bias for hydrophobic amino acids that seemed to impair correct predictions. Therefore, phage display-selected peptides may be over-hydrophobic and of high affinity, while natural interaction motifs are rather hydrophilic and mostly combine low affinity with high specificity. We suggest that potential amino acid composition bias should systematically be investigated when applying phage display data to the prediction of specific natural domain-linear motif interactions.     

颜氏大疣蛛蛛毒中多肽和蛋白质的多样性分析

对颜氏大疣蛛Macrothele yani蛛毒所富含的多肽与蛋白质多样性进行探索,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和超高效液相色谱-电喷雾-四极杆-飞行时间质谱技术分离和鉴定颜氏大疣蛛蛛毒中的蛋白质和多肽,并对其相对分子质量分布多样性进行分析。结果显示:粗毒中所含蛋白质的相对分子质量主要分布在35kDa以上。在17~135kDa分离度较佳的共有11条电泳条带,主要集中于40~120kDa附近;在75kDa附近弥散着高丰度的蛋白条带,75kDa以上的蛋白质最丰富。粗毒经色谱分离后得到超过50个色谱峰,经质谱鉴定得到121个物质成分,其中,多肽类物质的相对分子质量呈双峰式分布,21%分布在500~2000 Da,76%分布在3000~5000Da,为粗毒中多肽含量最丰富的部分,且集中于35~60min的保留时间内被洗脱。研究结果表明,颜氏大疣蛛蛛毒中含有较为丰富的多肽和蛋白类物质,这些物质的相对分子质量分布特征与已报道的其他蜘蛛既有相似性又存在具体差异。本文展示了颜氏大疣蛛蛛毒的分子多样性,为后续该毒素的物质基础研究及药用价值开发提供参考。     

脓毒症单核巨噬细胞特异性结合肽的筛选

利用噬菌体随机肽库展示技术,筛选出与脓毒症单核/巨噬细胞特异性结合的短肽,探索脓毒症治疗的新方法.分别以经过脂多糖(lipopolysaccharide, LPS)处理的人外周血单核细胞株(THP-1)细胞作为筛选的靶细胞,以未经LPS处理的THP-1细胞作为非特异性噬菌体吸附细胞,对噬菌体随机环七肽库进行4轮“差减"筛选,经过细胞ELISA验证阳性噬菌体克隆,对获得的阳性克隆进行DNA测序及生物信息学分析,并进一步利用免疫荧光实验,鉴定噬菌体克隆与LPS处理THP-1细胞的结合特异性.4轮筛选后,随机挑取的噬菌体克隆,测序后得到可与LPS处理的THP-1细胞特异性结合肽.对去冗余后的七肽进行Clustal W多序列比对分析和BlastP蛋白同源相似性分析,细胞免疫荧光检测确定获得的噬菌体展示七肽可与LPS处理的THP-1细胞特异性结合.噬菌体随机肽库技术为脓毒症单核/巨噬细胞表面靶位的筛选提供了高效、快捷的筛选体系,实验获得的多肽基序具有高度保守性和细胞特异性,这些多肽的生物活性将是下一步的研究内容.     

利用噬菌体表面展示技术筛选转铁蛋白黏附肽的研究

为了筛选转铁蛋白黏附肽,应用噬菌体表面展示技术经过三轮生物淘选,成功地从随机七肽库中得到黏附转铁蛋白的重组噬菌体克隆,经过相对亲和力常数测定和DNA测序得到4个转铁蛋白黏附肽的序列。实验中以回收率和选择比为操作参数,对淘选进行了优化,并发展了一种基于噬菌体滴度的相对亲和力常数测定方法。转铁蛋白受体是一种有效的肿瘤标记物,利用转铁蛋白为载体可以实现药物靶向运输,因此转铁蛋白黏附肽将是重组蛋白质药物连接转铁蛋白的有用标签。     

辅助噬菌体在噬菌体展示中的应用及研究进展

噬菌体展示技术是一种将外源肽或蛋白质与特定噬菌体衣壳蛋白相融合,展示于噬菌体表面来构建蛋白质或多肽文库,并从中筛选目的蛋白、多肽或抗体的基因工程高新技术。噬菌粒/辅助噬菌体系统是最常用的噬菌体展示系统,此系统中辅助噬菌体对噬菌粒的复制和组装发挥着至关重要的作用。本文结合当今该领域的最新研究动态,概述了噬菌粒和辅助噬菌体双基因组系统,着重介绍了不同辅助噬菌体的特点及其突变机制,并对其应用前景进行了展望,以期为该技术的进一步完善提供一定的借鉴作用。     

SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli

Antibody phage display is a key technology for the generation of recombinant (human) antibodies for research, diagnostics and therapy. Most antibody fragments can only be folded correctly in the oxidizing environment of the periplasm of Escherichia coli. A multitude of leader peptides has been used for secretion of antibody::pIII fusion proteins into the periplasm, but a systematic study of their impact on the performance of antibody phage display systems has not been reported so far. In this work we have analysed the influence of various leader peptides on antibody phage display efficiency and production yields of soluble antibody fragments. Four leader peptides using the Sec pathway (PelB, OmpA, PhoA and pIII) and three using the SRP pathway (DsbA, TorT and TolB) were compared. Both pathways are compatible with antibody phage display and the production of soluble antibody fragments. The applicability of the SRP pathway to antibody phage display and the production of functional scFvs is shown here for the first time.     

Identification of peptides that bind to the constant region of a humanized IgG1 monoclonal antibody using phage display

The pFc' fragments of a humanized IgG1 monoclonal antibody were generated by digestion with immobilized pepsin. These pFc' fragments were separated from F(ab')2 fragments by affinity chromatography. The pFc' fragments corresponding to the constant region of the humanized IgG1 monoclonal antibody were used as targets for phage display using variable-length peptide libraries. Interacting phage-displayed peptides were selected by repetitious cycles of target screening and phage amplification. Peptide sequences, deduced by sequencing DNA from isolated phage, were aligned and analyzed for amino acid motifs against each other and protein A. These results indicated that an amino acid motif has been identified using phage display technology that is sufficient for pFc' binding. Furthermore, the peptides derived from this study may prove useful in the development of peptidomimetic alternatives to protein A for use in affinity chromatography.     

Immunogenically fit subunit vaccine components via epitope discovery from natural peptide libraries

Antigenic peptides that bind pathogen-specific Abs are a potential source of subunit vaccine components. To be effective the peptides must be immunogenically fit: when used as immunogens they must elicit Abs that cross-react with native intact pathogen. In this study, antigenic peptides obtained from phage display libraries through epitope discovery were systematically examined for immunogenic fitness. Peptides selected from random peptide libraries, in which the phage-displayed peptides are encoded by synthetic degenerate oligonucleotides, had marginal immunogenic fitness. In contrast, 50% of the peptides selected from a natural peptide library, in which phage display segments of actual pathogen polypeptides, proved very successful. Epitope discovery from natural peptide libraries is a promising route to subunit vaccines.     

Peptide phage display in biotechnology and biomedicine

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors, etc.). Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nanomaterials.     

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.     

De novo identification of cell-type specific antibody-antigen pairs by phage display subtraction. Isolation of a human single chain antibody fragment against human keratin 14.

The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte-specific antigens from a phage display antibody repertoire by using phage display subtraction. Phage display is a method of displaying foreign molecules on the surface of filamentous bacteriophage particles. It allows the interaction between two cognate molecules to be analysed through affinity selections. Recently, large repertoires of phage displayed human antibody fragments have been constructed. From such repertoires, antibodies can be obtained in vitro without the need for immunization or the hybridoma technology. A novel subtractive strategy for selecting antibodies from phage libraries was applied. Phage antibodies were selected against immobilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand. A competing cell lysate was used to reduce retrieval of phage antibodies with specificities to commonly non-differentially expressed antigens. A monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identified as demonstrated by ELISA assays and immunoblotting analysis. The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotting based on 2D PAGE and a corresponding 2D PAGE protein database. In accordance with the expected tissue localization of K14, the identified scFv stained the basal layer of human epidermis by indirect immunofluorescence analysis. Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody-antigen pairs that characterize a specific cell type.