Neuronal affinity of a C7C loop peptide identified through phage display

Phage display is a promising tool for the screening of peptides with high affinity for specific cells. Here we describe a novel peptide with neuronal affinity isolated from a C7C library. We designed a two-tiered biopanning strategy initially selecting for ganglioside binding and subsequently selecting for binding to PC12 cells. At the completion of biopanning, 54.8% of phage clones bore the identical peptide (Tet.C7C.1). Immunofluorescence confirmed selective binding of this clone to differentiated PC12 cells. Tet.C7C.1 was synthesized and fluorescein conjugated. The synthetic peptide binds neuronal cell lines (SH-SY5Y, NSC-34 and PC12 cells) and tissue (DRG and spinal cord). The C7C structure creates a loop that minimizes the impact of peptide insertion on the confirmation of the recipient protein. Small loop peptides have the ideal characteristics for modification of viral vector capsids without undermining genome packaging. The neuronal binding properties of this peptide may be applied in the development of neurotropic viral vectors.     

Neuronal affinity of a C7C loop peptide identified through phage display

Phage display is a promising tool for the screening of peptides with high affinity for specific cells. Here we describe a novel peptide with neuronal affinity isolated from a C7C library. We designed a two-tiered biopanning strategy initially selecting for ganglioside binding and subsequently selecting for binding to PC12 cells. At the completion of biopanning, 54.8% of phage clones bore the identical peptide (Tet.C7C.1). Immunofluorescence confirmed selective binding of this clone to differentiated PC12 cells. Tet.C7C.1 was synthesized and fluorescein conjugated. The synthetic peptide binds neuronal cell lines (SH-SY5Y, NSC-34 and PC12 cells) and tissue (DRG and spinal cord). The C7C structure creates a loop that minimizes the impact of peptide insertion on the confirmation of the recipient protein. Small loop peptides have the ideal characteristics for modification of viral vector capsids without undermining genome packaging. The neuronal binding properties of this peptide may be applied in the development of neurotropic viral vectors.     

Linear mimotope analysis of Iranian cobra (Naja oxiana) snake venom using peptide displayed phage library

Snake venom pharmacologically comprises active components. This study aimed to identify mimotopes of Naja oxiana. Phage display peptide library, Ph.D.C7C, was utilized and biopanning carried out on immobilized horse antibodies. Three rounds of selection were performed on purified antibodies. Single phages randomly selected from the third round of biopanning were amplified and subjected to DNA sequencing. DNA sequencing results revealed 35 peptides with unique sequences. Obtained peptides represent some linear mimicking epitopes of venom antigens that involve in interaction with antibodies and could be used for further immunological and venomics study.     

抗人B-淋巴细胞刺激因子噬菌体单链抗体库的构建

目的构建抗人B-淋巴细胞刺激因子噬菌体单链抗体库。方法从B-淋巴细胞刺激因子免疫小鼠的脾细胞中抽提总RNA,经逆转录聚合酶反应合成cDNA,分别扩增小鼠重链可变区基因和轻链可变区基因。通过重叠延伸拼接法将重链可变区基因和轻链可变区基因组装成单链抗体基因,重组于载体pFUSE5,电转化E.coliXL 1-Blue。在辅助噬菌体援救下,构建成噬菌体单链抗体库。采用聚合酶链反应、核苷酸序列测定和酶联免疫分析鉴定单链抗体库的特征。结果本抗体库的噬菌体库容量约1×106,单链抗体基因插入效率为93%,存在序列差异性并含有抗B-淋巴细胞刺激因子单链抗体。结论成功地构建了抗人B-淋巴细胞刺激因子单链噬菌体抗体库,为筛选抗B-淋巴细胞刺激因子单链抗体药物奠定了良好的基础。     

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.     

Identification of new peptide ligands for epidermal growth factor receptor using phage display and computationally modeling their mode of binding

Peptide phage display, a powerful method for ligand identification, was used to identify new peptide ligands for epidermal growth factor receptor. A-431 cells expressing epidermal growth factor receptor were used as the matrix in a cell-based subtractive biopanning approach using a 7-mer peptide displaying phage library. Two novel peptide ligands were identified and tested for their affinities and functional effects on epidermal growth factor receptor. The identified peptides were able to inhibit the epidermal growth factor-induced phosphorylation of epidermal growth factor receptor in a concentration-dependent manner. The results of affinity binding experiments showed that the natural ligand, that is epidermal growth factor, was able to inhibit competitively the binding of peptide-bearing phage to epidermal growth factor receptor expressing A-431 cells. Molecular modeling studies were used to calculate the free energies for the binding of peptides to the receptor-binding site as well as proposing the interaction modes for this binding. The calculated values for the binding energies were found to be similar to our experimental data and those of previously reported studies.     

Selective targeting of a laccase from Stachybotrys chartarum covalently linked to a carotenoid‐binding peptide

Abstract: A two‐step targeting strategy was used to identify improved laccases for bleaching carotenoid‐containing stains on fabric. We first applied a modified phage display technique to identify peptide sequences capable of binding specifically to carotenoid stains and not to fabric. Prior deselection on the support on which the carotenoid was localized, increased stringency during the biopanning target selection process, and analysis of the phage peptides’ binding to the target after acid elution and polymerase chain reaction (PCR) postacid elution, were used to isolate phage peptide libraries with increased binding selectivity and affinity. Peptide sequences were selected based on identified consensus motifs. We verified the enhanced carotenoid‐binding properties of the peptide YGYLPSR and subsequently cloned and expressed C‐terminal variants of laccase from Stachybotrys chartarum containing carotenoid‐binding peptides YGYLPSR, IERSAPATAPPP, KASAPAL, CKASAPALC, and SLLNATK. These targeted peptide–laccase fusions demonstrate enhanced catalytic properties on stained fabrics.     

Identification of oligopeptide binding to colon cancer cells separated from patients using laser capture microdissection

The development of intravascular conjugates that efficiently deliver genes or drugs to tumors is limited by the lack of efficacious targeting ligands. Small targeting peptides, such as those iterated by phage display technology, offer enormous potential for these applications. The majority of reports published to date have focused on the identification of peptides isolated for their ability to bind to human cancer cell lines in vitro, and have failed to account for the loss of polarization and de-differentiation of such cells from their in vivo state. Here, we report a novel approach for the identification of peptides capable of binding specifically to cancer cells derived from clinically resected human colon cancer. In this strategy, laser capture microdissection (LCM) is performed on a surgically resected colon cancer specimen to separate only cancer cells from the specimen. Subsequently, biopanning was performed on the LCM-selected colon cancer cells to identify peptide sequences that bound specifically to them. A peptide containing the SPT motif was selected as the most promising consensus sequence binding specifically to the LCM-selected colon cancer cells. Phage clones displaying the SPT motif demonstrated 9-fold higher binding to colon cancer cells derived from a patient than insertless phage (p < 0.05), while, recovery of the SPT phage from the colon cancer cell lines DLD-1 and HCT-15 was 7-fold higher than that of the control insertless phage (p < 0.05). The binding of SPT phage to colon cancer cells from the patient was confirmed by immunofluorescence. Additionally, a synthesized SPT-containing peptide (SPTKSNS) showed binding activity in the absence of mitogenic effects on colon cancer cells in vitro. In summary, we have introduced LCM into a biopanning procedure and identified a small peptide that binds preferentially to colon cancer cells derived from a clinically resected sample. This procedure could be applicable for the design of customized cancer cell targeting methodologies using clinical biopsy samples from human subjects.     

尖吻蝮蛇毒研究进展

尖吻蝮(又名五步蛇,中药材名为蕲蛇)蛇毒,是从尖吻蝮毒腺中分泌的毒液,内含磷脂酶A₂、丝氨酸蛋白酶、金属蛋白酶、C型凝集素、L-氨基酸氧化酶等多种蛋白类成分和肽类成分,具有多种生物活性,在抗肿瘤、抗血栓、抗炎、抗菌等方面发挥着重要作用。近年来,蛇毒研究日益广泛,但目前仍缺乏对尖吻蝮蛇毒全面系统的研究。本文通过检索尖吻蝮蛇毒的相关研究进展,在其来源、鉴别、活性成分、毒性研究及质量研究等方面进行总结与分析,以期为尖吻蝮蛇毒进一步开发利用提供参考。     

噬菌体随机肽库淘选肺癌细胞NCI-H1299特异性结合多肽

目的利用噬菌体展示肽库筛选出与肺癌NCI-H1299细胞特异性结合的多肽,鉴定其特异性和亲和力,为寻找肺癌早期诊断和治疗标志物打下基础。方法以肺癌细胞NCI-H1299为靶细胞,肺二倍体成纤维细胞MRC-5为吸附细胞,在37℃下对噬菌体随机十二肽库进行三轮减性筛选,挑取单克隆ELISA初步鉴定噬菌体克隆亲和力;阳性克隆提取质粒,测序后合成多肽,鉴定多肽的亲和力及特异性。结果经ELISA初步鉴定,随机挑选的20个单克隆中,其中6号对NCI-H1299亲和力最高,将其命名为PhageZS6。细胞免疫荧光试验进一步证明,合成的相应多肽FITCZS6对NCI-H1299具有高亲和力。结论利用噬菌体随机十二肽成功筛选出与肺癌细胞具有较高亲和力的多肽ZS6,为肺癌的早期诊断和靶向治疗奠定基础。     

Attaching the phage display-selected GLA peptide to liposomes: factors influencing target binding

In our previous study, phage display selections were performed by in situ perfusion of a random peptide library through a mouse brain. This yielded two peptides (GLA and GYR) that showed significant binding to human brain endothelial cells (hCMEC/D3) when displayed on phage particles, but not to human umbilical vein endothelial cells (HUVECs). In the present study, these peptides were produced synthetically and coupled to liposomes to investigate the capacity of the peptides to act as ligands for targeting to hCMEC/D3 cells. Flow cytometry studies showed that these peptides when coupled to liposomes showed weak binding to the target brain endothelial cells. We hypothesized that the weak endothelial cell binding of the selected peptides when coupled to liposomes as compared to the binding of the peptides displayed on phage particles may be ascribed to: change of vehicle shape, change of peptide density, or change of peptide conformation. Peptide density on the liposomes influenced binding of the liposomes to the cells, however, this effect was minor. To study the influence of the peptide conformation, the GLA peptide was recombinantly produced fused to the N1-N2 domains of the phage p3 minor coat protein (p3-GLA) to mimic its conformation when displayed on phage particles. Binding of liposomes modified with either the GLA peptide or the p3-GLA protein to hCMEC/D3 cells was studied, and the p3-GLA-liposomes showed a higher binding to the cells compared to the GLA-liposomes. The experiments demonstrate that bringing the GLA peptide into the original phage protein environment restores and improves the peptide binding capacity and suggest that the GLA peptide, with some modifications, may be used as a brain-targeting ligand in the future.     

Use of a Phage Display Library to Identify Oligopeptides Binding to the Lumenal Surface of Polarized Endothelium by Ex Vivo Perfusion of Human Umbilical Veins

Human endothelial-specific targeting peptides were identified by biopanning within freshly-obtained human umbilical cords. Umbilical veins were cleaned in situ and M13 phage display libraries were passed through the cords. Tightly bound phage were recovered following isolation of endothelial cells by collagenase digestion and homogenisation, allowing production of enriched phage libraries for subsequent rounds of panning. After five rounds of biopanning, five promising sequences were selected and the binding of the corresponding phage clones was compared in perfused umbilical veins. Each of these peptides showed substantial binding, although the clone encoding the heptapeptide KPSGLTY showed the greatest, some 89-times greater than insertless phage. Binding of this phage clone was examined to cells in vitro, where it demonstrated at least five-times greater binding to isolated human umbilical vein endothelial cells than to 911, SKOV3, B16F10 and Cos7 cells. These initial peptides may prove useful targeting agents for endothelial-selective delivery, and this powerful approach should be readily applicable to biopanning in a broad range of human vessels ex vivo.     

Cyclic Peptides as Therapeutic Agents and Biochemical Tools

There are many cyclic peptides with diverse biological activities, such as antibacterial activity, immunosuppressive activity, and anti-tumor activity, and so on. Encouraged by natural cyclic peptides with biological activity, efforts have been made to develop cyclic peptides with both genetic and synthetic methods. The genetic methods include phage display, intein-based cyclic peptides, and mRNA display. The synthetic methods involve individual synthesis, parallel synthesis, as well as split-and-pool synthesis. Recent development of cyclic peptide library based on split-and-pool synthesis allows on-bead screening, in-solution screening, and microarray screening of cyclic peptides for biological activity. Cyclic peptides will be useful as receptor agonist/antagonist, RNA binding molecule, enzyme inhibitor and so on, and more cyclic peptides will emerge as therapeutic agents and biochemical tools.     

Development of a Monoclonal scFv against Cytotoxin to Neutralize Cytolytic Activity Induced by Naja atra Venom on Myoblast C2C12 Cells

The Taiwanese cobra, Naja atra, is a clinically significant species of snake observed in the wild in Taiwan. Victims bitten by N. atra usually experience severe pain and local tissue necrosis. Although antivenom is available for treatment of cobra envenomation, its neutralization potency against cobra-induced necrosis is weak, with more than 60% of cobra envenoming patients developing tissue necrosis after antivenom administration. The present study found that cytotoxin (CTX) is a key component of N. atra venom responsible for cytotoxicity against myoblast cells. Anti-CTX IgY was generated in hens, and the spleens of these hens were used to construct libraries for the development of single chain variable fragments (scFv). Two anti-CTX scFv, S1 and 2S7, were selected using phage display technology and biopanning. Both polyclonal IgY and monoclonal scFv S1 reacted specifically with CTX in cobra venom. In a cell model assay, the CTX-induced cytolytic effect was inhibited only by monoclonal scFv S1, not by polyclonal IgY. Moreover, the neutralization potency of scFv S1 was about 3.8 mg/mg, approximately three times higher than that of conventional freeze-dried neurotoxic antivenom (FNAV). Collectively, these results suggest that scFv S1 can effectively neutralize CTX-induced cytotoxicity and, when combined with currently available antivenom, can improve the potency of the latter, thereby preventing tissue damage induced by cobra envenoming.     

Development of diagnostically and therapeutically useful human antibody medicines by phage display system

Phage display has been utilized for making recombinant antibody fragments (Fab or single chain Fv) of human, mouse, or other origins. After construction of an antibody combinatorial library, antigen-specific recombinant antibody fragments can be easily isolated by biopanning of the phage library displaying antibody fragment fused with viral coat protein III against antigen proteins, antigen-expressing live cells, or fixed cells. Using this technique, a variety of human recombinant antibody fragments can be retrieved from bone marrow cells, lymph node cells, or peripheral blood cells of patients with infectious diseases, autoimmune diseases, and cancer. To develop diagnostically and therapeutically useful human antibody medicines, we should first select recombinant antibody fragments not only with antigen-binding activity but also with bioactivity such as virus or toxin neutralization, or tumor-specific cytotoxicity. To achieve this goal, several steps in antibody phage display may be improved: 1) a larger library should be constructed for possible isolation of minor populations present in the repertoire; 2) the biopanning procedure should be improved for isolation of antibody fragments reactive with immunologically minor epitopes; 3) the screening procedure should be based on the measurement of the bioactivity as well as the antigen-binding activity; 4) if necessary, the affinity and specificity of selected antibody fragments should be improved. In this review, I discuss how to isolate clinically useful recombinant antibody fragments efficiently using a phage display system introducing our achievements.     

晚期糖基化终产物受体胞外段的原核表达及其相互作用蛋白的筛选

目的构建晚期糖基化终产物受体(receptor for ad-vanced glycation end-product,RAGE)胞外段基因的原核表达载体并诱导表达,应用噬菌体展示技术筛选其相互作用蛋白。方法用RT-PCR方法扩增人RAGE胞外段cDNA,构建于原核表达载体pET14b,转化大肠杆菌BL21,IPTG诱导RAGE胞外段融合蛋白表达,用Ni2+-NTA亲和层析柱进行纯化并行Western blot鉴定。以重组RAGE胞外段蛋白为靶分子,进行3轮噬菌体展示环七肽库的筛选,从第3轮洗脱物中随机挑选分隔良好的噬菌体克隆扩增后进行ELISA鉴定。对获得的阳性噬菌体克隆分别进行扩增、纯化,提取DNA测序后翻译为氨基酸序列,用BLAST软件搜索Gen-Bank中的同源序列。结果成功构建并表达、纯化了RAGE胞外段融合蛋白。经过3轮筛选后,随机挑取的24个噬菌体克隆中11个可与RAGE胞外段结合。对11个噬菌体测序后用BLAST软件搜索同源序列,得到14个编码蛋白。结论应用噬菌体展示技术筛选到与RAGE胞外段相互作用的蛋白,为进一步研究其功能和作用机制提供新线索。     

Identification and characterization of peptide probes directed against PKCα conformations

Abstract: Phage display is a powerful technology that allows identification of high affinity peptides that bind specifically to a given molecular target. Using a highly complex peptide display library, we have identified separate classes of peptides that bind to protein kinase C alpha (PKCα) only under activation conditions. Furthermore, peptide binding was specific to PKCα and not to any of the other closely related PKC isoforms. The conformational and isoform specificity of the peptide binding was demonstrated using surface plasmon resonance as well as time‐resolved fluorescence assays. Kinase assays showed that these peptides were not direct substrates for PKC nor did they inhibit phosphorylation of PKC substrates. These peptides are most likely directed against protein–protein interaction sites on PKC. The data presented here offers another example of application of phage display technology to identify conformation‐dependent peptide probes against therapeutically important drug targets. These peptides are ideally suited to be used as surrogate ligands to identify compounds that bind specifically to PKCα, as well as conformational probes to detect activated forms of PKCα.     

Use of a phage display library to identify oligopeptides binding to the lumenal surface of polarized endothelium by ex vivo perfusion of human umbilical veins

Human endothelial-specific targeting peptides were identified by biopanning within freshly-obtained human umbilical cords. Umbilical veins were cleaned in situ and M13 phage display libraries were passed through the cords. Tightly bound phage were recovered following isolation of endothelial cells by collagenase digestion and homogenisation, allowing production of enriched phage libraries for subsequent rounds of panning. After five rounds of biopanning, five promising sequences were selected and the binding of the corresponding phage clones was compared in perfused umbilical veins. Each of these peptides showed substantial binding, although the clone encoding the heptapeptide KPSGLTY showed the greatest, some 89-times greater than insertless phage. Binding of this phage clone was examined to cells in vitro, where it demonstrated at least five-times greater binding to isolated human umbilical vein endothelial cells than to 911, SKOV3, B16F10 and Cos7 cells. These initial peptides may prove useful targeting agents for endothelial-selective delivery, and this powerful approach should be readily applicable to biopanning in a broad range of human vessels ex vivo.     

Creation of novel cell-penetrating peptides for intracellular drug delivery using systematic phage display technology originated from Tat transduction domain

Many biologically active proteins need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm. Cell penetrating peptides (CPPs) have been developed to efficiently deliver a wide variety of cargo in a fully biological active form into a range of cell types for the treatment of multiple preclinical disease models. To further develop this methodology, we established a systematic approach to identify novel CPPs using phage display technology. Firstly, we screened a phage peptide library for peptides that bound to the cell membrane. Secondly, to assess functionality as intracellular carriers, we recombined cDNAs of binding peptides with protein synthesis inhibitory factor (PSIF) to create fusion proteins. Randomly chosen clones were cultured and expression of peptide-PSIF fusion proteins induced, followed by screening of protein synthesis activity in cells. Using this systematic approach, novel and effective CPPs were rapidly identified. We suggest that these novel cell-penetrating peptides can utilized as drug delivery tools for protein therapy or an analytical tool to study mechanisms of protein transduction into the cytoplasm.