Analysis of the Glycyrrhizic Acid-Binding Sites with Phage Display

Phages that expose peptides specifically interacting with glycyrrhizic acid (GA) were selected from a phage peptide library by affinity selection and ELISA. Amino acid sequence analysis of the selected peptides and human proteins with the SIM program revealed homology to tyrosine protein kinases, serine/threonine protein kinases, tyrosine phosphatases, and some receptors. Analysis of the peptide and virus protein sequences with the BLAST program showed that GA has affinity for various surface proteins of several human viruses such as HIV-1, hepatitis C virus, and herpesviruses.     

Determination of glycyrrhizic acid binding sites by a phage display method

Phages that expose peptides specifically interacting with glycyrrhizic acid (GA) were selected from a phage peptide library by affinity selection and ELISA. Amino acid sequence analysis of the selected peptides and human proteins with the SIM program revealed homology to tyrosine protein kinases, serine/threonine protein kinases, tyrosine phosphatases, and some receptors. Analysis of the peptide and virus protein sequences with the BLAST program showed that GA has affinity for various surface proteins of several human viruses such as HIV-1, hepatitis C virus, and herpesviruses.     

Selection and characterization of colorectal cancer cell-specific peptides

To develop a novel peptide probe for imaging detection of colorectal cancer, a 12-mer phage display library was used to select peptides that bind specifically to the human colorectal cancer cell line Caco-2. After four rounds of panning, four phage clones that bound specifically to the Caco-2 cells were selected. The phage clone SP-2 had a particularly high affinity and specificity for Caco-2 cells. This clone was identified using a series of methods. The peptide SP-2 that was displayed on phage SP-2 exhibited a high specificity to Caco-2 cells. Thus, the peptide SP-2 could be a candidate probe for the detection of colorectal cancer.     

Selection and Characterization of a Peptide Specifically Targeting to Gastric Cancer Cell Line SGC-7901 Using Phage Display

Peptides selected from phage display have great potential to become probes for the imaging detection of the cancer. To develop the peptide probe for diagnosis of GC, a 12-mer phage display library was used to select peptides that bind specifically to the human GC cell line SGC-7901. After four rounds of in vitro selection, five phage clones that bound specifically to the SGC-7901 cells were selected. The phage clone GP-5 had a particularly high affinity and specificity for SGC-7901 cells. This clone was identified using a series of methods. The peptide GP-5 that was displayed on phage GP-5 exhibited high specificity to SGC-7901 cells and gastric tissues. Thus, the peptide GP-5 displays excellent potential for imaging detection of human gastric cancer.     

Screening and Identification of a Specific Binding Peptide to Ovarian Cancer Cells from a Phage-Displayed Peptide Library

To select specific binding peptides for imaging and detection of human ovarian cancer. The phage 12-mer peptide library was used to select specific phage clones to ovarian cancer cells. After four rounds of biopanning, the binding specificity of randomly selected phage clones to ovarian cancer cells was determined by enzyme-linked immunosorbent assay (ELISA). DNA sequencing and homology analysis were performed on specifically bound phages. The binding ability of the selected peptides to SKOV3 cells was confirmed by fluorescence microscopy and flow cytometry. After four rounds of optimized biological panning, phage recovery was 34-fold higher than that of the first round, and the specific phage clones bound to SKOV3 cells were significantly enriched. A total of 32 positive phage clones were preliminarily identified by ELISA from 54 randomly selected clones, and the positive rate was 59.3%. S36 was identified as the clone with best affinity to SKOV3 cells via fluorescence microscopy and flow cytometry. A representative clone of OSP2, S36 is expected to be an effective probe for diagnosis and treatment of ovarian cancer.

     

Identification by phage display selection of a short peptide able to inhibit only the strand transfer reaction catalyzed by human immunodeficiency virus type 1 integrase

Human immunodeficiency virus type 1 integrase catalyzes the integration of proviral DNA into the infected cell genome, so it is an important potential target for antiviral drug design. In an attempt to search for peptides that specifically interact with integrase (IN) and inhibit its function, we used an in vitro selection procedure, the phage display technique. A phage display library of random heptapeptides was used to screen for potential peptide ligands of HIV-1 IN. Several phage clones were identified that specifically bound IN. Two of the selected peptides (FHNHGKQ and HLEHLLF) exhibited a high affinity for IN and were chemically synthesized. High affinity was confirmed by a displacement assay which showed that these two synthetic peptides were able to compete with the phages expressing the corresponding peptide. These agents were assayed on the in vitro IN activities. While none of them inhibited the 3'-processing reaction, the FHNHGKQ peptide was found to be an inhibitor of the strand transfer reaction. Despite its high affinity for IN, the HLEHLLF peptide selected and assayed under the same conditions was unable to inhibit this reaction. We showed that the FHNHGKQ peptide inhibits specifically the strand transfer activity by competing with the target DNA for binding to IN. These IN-binding agents could be used as a base for developing new anti-integrase compounds as well as for structural studies of the still unknown three-dimensional structure of the entire integrase molecule.     

脑膜炎大肠杆菌IbeA蛋白结合肽序列的亲和筛选

应用噬菌体展示肽库技术,以重组的脑膜炎大肠杆菌致病蛋白IbeA作为靶分子,经过吸附-洗脱-扩增-再吸附的亲和筛选,随机挑选亲和力强的噬菌体克隆,进行ELISA、竞争抑制实验和序列测定。结果显示,经3轮淘选后,间接ELISA鉴定得到高亲和性结合IbeA蛋白的15个阳性克隆。竞争抑制实验结果表明,游离IbeA蛋白能竞争抑制噬菌体结合肽克隆与固相包被的IbeA蛋白的结合,其抑制作用随游离IbeA蛋白浓度的降低而减弱。测序结果得到5种阳性噬菌体克隆展示肽序列。上述结果提示以脑膜炎大肠杆菌IbeA蛋白为靶筛选所获得的噬菌体12肽克隆,具有特异性,其结合肽序列呈现相对保守性。建立的从噬菌体随机肽库筛选IbeA蛋白结合肽的方法具有方便、灵活和高效可行的特点。     

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.     

Isolation of multiple cell-binding ligands from different phage displayed-peptide libraries

A technical challenge in the development of biosensor devices for cancer detection and diagnosis is the identification of ligands that recognize cancer cells with high affinity and specificity. Furthermore, it is unlikely that one cell-binding ligand will provide sufficient biological information, thus, multiple ligands for a given cancer type will be needed for confident clinical diagnosis. Biopanning of phage displayed peptide libraries is a route to isolation of specific cell-binding reagents. A potential approach towards isolation of multiple ligands for a single cell type is to pan against the same cell type using different peptide libraries. Here we report the synthesis of a new 20-mer peptide-phage library and its use to select a peptide that binds to the large cell lung carcinoma cell line, H1299. The isolated phage clone binds H1299 cells 80 times better than a control phage and can distinguish between H1299 and normal control cells. The phage clone also binds to the lung pleura epidermoid cell line, Calu-1 but not to all lung carcinoma cell lines. The peptide is functional outside the context of the phage and tetramerization of the peptide on a trilysine core improves the affinity of the peptide. The tetrameric peptide can be used to deliver a fluorescent quantum dot to H1299 cells. Unexpectedly, the peptide shares sequence similarity to a previously isolated H1299-binding peptide isolated from a different 20-mer peptide library. Data suggests that the two peptides target the same cellular receptor. Our results imply that cell-based biopanning can isolate cell-binding ligands that may be of utility for cancer diagnosis, and isolation of cell-targeting peptides from different peptide libraries can expand the repertoire of cell-binding reagents.     

Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.     

Investigation and molecular mimicry of the antigen involved in the interaction between the monoclonal antibody 5D10 and the human breast cancer cell line MCF-7

Monoclonal antibody (mAb) 5D10 is directed against the human breast cancer cell line MCF-7. Biochemical characterization of the antibody epitope was attempted and revealed a complex, most likely carbohydrate-linked nature, which prevented isolation and further studies of the interaction. A major goal of this work was to generate structural mimics of the 5D10 epitope to serve as putative substitutes in such studies. A peptide library displayed on filamentous phage was used to select for mimotope peptide sequences. All positive phage clones selected from the library displayed the amino acid sequence H(2)N-QMNPMYYR-CO(2)H. This peptide sequence, as well as a branched form of the peptide, was found to bind mAb 5D10. Moreover, both peptide sequences were able to inhibit the binding of 5D10 to the MCF-7 cells in a concentration-dependent manner, with an EC(50) value in the range of 65 microM. According to these results, random phage peptide libraries can serve to identify mimotopic peptides for unknown complex cell surface epitopes.     

Peptides binding to a Gb3 mimic selected from a phage library

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.     

Design of a PKCδ-specific small peptide as a theragnostic agent for glioblastoma

Glioblastoma is an aggressive malignant brain tumor that starts in the brain or spine and frequently recurs after anticancer treatment. The development of an accurate diagnostic system combined with effective cancer therapy is essential to improve prognosis of glioma patients. Peptides, produced from phage display, are attractive biomolecules for glioma treatment because of their biostability, nontoxicity, and small size. In this study, we employed phage display methodology to screen for peptides that specifically recognize the target PKCδ as a novel biomarker for glioma. The phage library screening yielded four different peptides displayed on phages with a 20- to 200-pM K_d value for the recombinant PKCδ catalytic domain. Among these four phage peptides, we selected one to synthesize and tagged it with fluorescein isothiocyanate (FITC) based on the sequence of the PKCδ-binding phage clone. The synthetic peptide showed a relative binding affinity for antibody and localization in the U373 glioma cell. The kinase activity of PKCδ was inhibited by FITC-labeled peptide with an IC₅₀ of 1.4 μM in vitro. Consequently, the peptide found in this study might be a promising therapeutic agent against malignant brain tumor.     

细胞间粘附分子1特异结合肽的筛选及其生物功能

采用两种方法对噬菌体展示随机十五肽库进行亲和淘选 .ELISA法筛选特异结合高亲和力的阳性噬菌体单克隆 ,测序 ,得到 6个与人细胞间粘附分子 1(ICAM 1)高亲和力的噬菌体展示十五肽单克隆 .再经ELISA法从这 6个噬菌体单克隆中选择与ICAM 1亲和力最高的单克隆 ,同时利用蛋白空间结构位象模拟技术对小肽与ICAM 1的亲和力进行模拟研究 .最终获取目的小肽的氨基酸序列为GRGEFRGRDNSVSVV .目的单克隆噬菌体与ICAM - 1的亲和常数Ka 为 7 87× 10 7L mol .体外合成、纯化并标记目的小肽 .ELISA法验证目的小肽与人ICAM 1的结合呈浓度依赖性 ,抗ICAM 1多抗不能拮抗目的小肽与ICAM 1的结合 .采用免疫组化方法证实 ,此目的小肽具有与炎症组织中高表达的ICAM 1特异性结合的功能 .在动物体内 ,荧光标记的目的小肽具有向高表达ICAM 1的炎症部位特异性聚集的功能 .说明此目的肽可尝试作为以ICAM 1为靶的“肽导向药物”的前导肽 .     

从噬菌体随机肽库中筛选流感病毒神经氨酸酶的抑制多肽

目的:从噬菌体呈现12肽库中筛选与流感病毒神经氨酸酶特异性结合的肽。方法:以甲三型流感病毒裂解疫苗原液为靶分子,经过3轮生物淘选,从噬菌体随机肽库中筛选与之结合的噬菌体。用ELISA方法鉴定噬菌体克隆与靶分子的结合力,用荧光方法测定噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶的抑制活性。对筛选到的阳性克隆进行DNA序列测定并推导出相应的氨基酸序列。结果:经过3轮筛选后,42个噬菌体克隆与靶分子有高度亲和力,23个噬菌体克隆对流感病毒A/Sydney/5/97(H3N2)神经氨酸酶有抑制活性。对27个噬菌体克隆的测序结果表明,分别有10个和2个克隆的序列是一致的,其氨基酸序列分别为KSLSRHDHIHHH和WPRHHHSASVQT。结论:通过噬菌体肽库筛选到抑制流感病毒神经氨酸酶的12肽,为进一步研究对流感病毒神经氨酸酶有抑制活性的分子药物奠定了基础。     

Phage-display screening identifies LMP1-binding peptides targeting the C-terminus region of the EBV oncoprotein

Latent membrane protein 1 (LMP1), a major oncoprotein of Epstein Barr Virus (EBV) is responsible for transforming B lymphocytes in vitro. LMP1 is overexpressed in several EBV-associated malignancies, and different approaches have been developed to reduce its level and accordingly its oncogenic function in tumor tissues. This study aimed to use phage display peptide library to obtain peptides which could specifically bind to the cytoplasmic region of LMP1 to prevent its interaction with signaling proteins. The LMP1 C-terminus region was produced in bacterial E. coli and used as target for the phage library panning. After 3 rounds, 20 phage clones were randomly selected and 8 showed high binding affinity to the recombinant C-terminus LMP1 protein. The most interesting candidates are the FO5 “QPTKDSSPPLRV” and NO4 “STTSPPAVPHNN” peptides since both bind the C-terminus LMP1 as showed by molecular docking. Furthermore, sequence alignment revealed that the FO5 peptide shared sequence similarity with the Death Receptor 4 which belongs to the tumor necrosis factor-related apoptosis-inducing receptor which plays key role in anti-tumor immunity.     

核受体PPARγ结合小肽的筛选及其功能

为筛选与核受体过氧化物酶体增殖物激活受体γ(PPARγ)结合的功能短肽,在大肠杆菌BL21(DE3)中表达PPARγ配体结合域(LBD)的融合蛋白,并利用Ni2+-NTA离子交换树脂对表达蛋白进行纯化.以此纯化蛋白为靶,采用固体包被法对噬菌体展示随机十二肽库及环七肽库进行亲和筛选.经ELISA法鉴定特异结合的高亲和力阳性噬菌体单克隆并测序.同时利用PPARγ的配体rosiglitazone与噬菌体小肽进行竞争性结合抑制实验.最终获得与PPARγ-LBD高亲和力的十二肽3个,环七肽5个,分别含LXXLL和DXXRW(其中X为非特异氨基酸残基)保守序列.Rosiglitazone不影响噬菌体小肽与靶蛋白的结合,说明获得与配体rosiglitazone结合位点不同的目的肽.     

Selection of a phage-displayed peptide recognized by monoclonal antibody directed blocking the site of hepatitis C virus E2 for human CD81

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). HCV envelope glycoprotein 2 (E2) most likely plays a pivotal role in binding to host cells by interacting with the hCD81 molecule. In this study, a phage-displayed peptide library was used to select small peptides with anti-hCD81 monoclonal antibody JS-81. The output/input ratio of phages increased about 91 fold after the third round of selection. Eight of the 30 phage clones selected from the phage library showed specific binding to the anti-hCD81 by enzyme linked immunosorbent assay (ELISA). Competitive inhibition test further demonstrated that HCV E2 could significantly inhibit the binding of a positive phage clone to anti-hCD81 JS-81. Exogenous small peptide ATWVCGPCT contained by the positive phage clones showed aligned with the hCD81 sequence from 153-161 by sequence analyses. These results suggest that the selected ATWVCGPCT is a novel hCD81-like small peptide, which can block the binding site of HCV E2 for hCD81. It may be of further application on development of antiviral agents targeting the stage of HCV entry.     

Potential application of antibody-mimicking peptides identified by phage display in immuno-magnetic separation of an antigen

Phage display was performed against human IgG (hIgG) through five rounds of 'biopanning'. Each round consisted of: (1) incubating a library of phage-displayed 12-mer peptides sequences on hIgG-coated magnetic beads, (2) washing the unbound phages, and (3) eluting the bound phages. The eluted phages were either amplified to enrich the pool of positive clones or subjected to the next round without amplification. Through ELISA, four clones (F9, D1, G5, and A10) showing specific binding affinity to hIgG were identified. Among these, F9 had the highest affinity (K(d)=6.2nM), only one order of magnitude lower than the native anti-hIgG antibody (0.66nM). Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Among them, D1 peptide showed the highest binding affinity to hIgG via SPR biosensor measurements. This peptide was conjugated to biofunctionalized magnetic beads, and its immuno-binding ability was compared with that of the native antibody immobilized to magnetic beads. The mol-to-mol binding efficacy of the peptide-coated magnetic beads was approximately 1000-fold lower than that of the antibody-coated magnetic beads. Our results suggest a feasibility of using antibody-mimicking peptides identified by phage display technique for immuno-magnetic separation of an antigen.     

抗松材线虫纤维素酶单链抗体库的构建及筛选

构建鼠源性松材线虫纤维素酶（Bursaphelenchus xylophilus cellulase, BXC）的噬菌体单链抗体库,从中筛选特异性BXC的单链抗体。以BXC为抗原免疫BALB/C小鼠,从脾脏提取总RNA,用RT-PCR技术扩增小鼠抗体重链（V_H）和轻链(V_L)可变区基因。经重叠PCR(SOE-PCR)在体外将V_H和V_L连接成单链抗体(scFv)基因,并克隆到噬菌粒载体pCANTAB5E中,电转化至大肠杆菌TG1,经辅助噬菌体超感染,成功构建了库容为5×10⁴的Anti-BXC单链抗体库,并从该抗体库中初步筛选到了特异性识别BXC的噬菌体单链抗体scFv。将表面展示单链抗体的单克隆噬菌体转化大肠杆菌HB2151进行可溶性表达,SDS-PAGE及Western blot分析结果显示,可溶性scFv获得表达,且与BXC具有结合活性,为松材线虫的检验检疫以及病理学研究奠定了基础。