神经生长因子低亲和力受体(p75NTR)的模拟配基的筛选

人神经生长因子低亲和力受体 (p75NTR)转染R2细胞而建立的R2L1细胞 ,在去血清培养时发生凋亡 ,该作用可被神经生长因子 (NGF)所抑制 .用R2L1和R2两种细胞差式筛选噬菌体随机 7肽库和 1 2肽库 ,获得和p75NTR特异结合的噬菌体 .测定DNA序列后得到有关多肽的氨基酸序列 .7肽库共有序列为C (H D)LP(K M)HPM C ;1 2肽库优势序列为TLPSPLALLTVH .化学合成相应的 2个短肽 .用细胞结合法和ELISA方法证实阳性噬菌体和合成短肽能与p75NTR结合 ,并证实了它们对R2L1细胞去血清培养后的凋亡有抑制作用     

Targeting phosphatidylserine on apoptotic cells with phages and peptides selected from a bacteriophage display library

Phosphatidylserine (PS) is a well-characterized biomarker for apoptosis. Ligands that bind to PS can be used for noninvasive imaging of therapy-induced cell death, particularly apoptosis. In this study, we screened a random 12-mer peptide phage library on liposomes prepared from PS. One clone displaying the peptide SVSVGMKPSPRP (designated as PS3-10) bound to PS approximately 4-fold better than its binding to phosphatidylcholine and 18-fold better than to bovine serum albumin in a solid-phase binding assay. In addition, the binding of the corresponding PS3-10 peptide to PS was significantly higher than that of a scrambled peptide. PS3-10 phages, but not a control 4-2-2 phage, bound to aged red blood cells that had PS exposed on their surface. Binding of PS3-10 phages and PS3-10 peptide to TRAIL-induced apoptotic DLD1 cells was 3.2 and 5.4 times higher than their binding to untreated viable cells, respectively. Significantly, immunohistochemical staining confirmed selective binding of PS3-10 phages to apoptotic cells. Our data suggest that panning of phage display libraries may allow the selection of suitable peptide ligands for apoptotic cells and that PS3-10 peptide may serve as a template for further development of molecular probes for in vitro and in vivo imaging of apoptosis.     

从噬菌体文库中筛选潜在的GMCSF的拮抗剂(英文)

以粒细胞巨噬细胞集落刺激因子（ＧＭＣＳＦ） 为筛选文库的靶分子, 通过高效筛选（Ｈｉｇｈ ｔｈｒｏｕｇｈｐｕｔｓｃｒｅｅｎｉｎｇ, ＨＴＳ） 方法来筛选多种多肽噬菌体文库, 在一个以噬菌体主要蛋白质为载体的多肽噬菌体文库中筛选到了一些与ＧＭＣＳＦ结合的多肽, 并通过了ＥＬＩＳＡ和微淘选（ｍｉｃｒｏｐａｎｎｉｎｇ） 实验的证实。这些多肽先导化合物经过进一步的优化, 可能成为ＧＭＣＳＦ细胞因子的拮抗剂     

New insights into the possible role of bacteriophages in host defense and disease

BACKGROUND: While the ability of bacteriophages to kill bacteria is well known and has been used in some centers to combat antibiotics - resistant infections, our knowledge about phage interactions with mammalian cells is very limited and phages have been believed to have no intrinsic tropism for those cells. PRESENTATION OF THE HYPOTHESIS: At least some phages (e.g., T4 coliphage) express Lys-Arg-Gly (KGD) sequence which binds beta3 integrins (primarily alphaIIbbeta3). Therefore, phages could bind beta3+ cells (platelets, monocytes, some lymphocytes and some neoplastic cells) and downregulate activities of those cells by inhibiting integrin functions. TESTING THE HYPOTHESIS: Binding of KGD+ phages to beta3 integrin+ cells may be detected using standard techniques involving phage - mediated bacterial lysis and plaque formation. Furthermore, the binding may be visualized by electron microscopy and fluorescence using labelled phages. Binding specificity can be confirmed with the aid of specific blocking peptides and monoclonal antibodies. In vivo effects of phage - cell interactions may be assessed by examining the possible biological effects of beta3 blockade (e.g., anti-metastatic activity). IMPLICATION OF THE HYPOTHESIS: If, indeed, phages can modify functions of beta3+ cells (platelets, monocytes, lymphocytes, cancer cells) they could be important biological response modifiers regulating migration and activities of those cells. Such novel understanding of their role could open novel perspectives in their potential use in treatment of cardiovascular and autoimmune disease, graft rejection and cancer.     

利用噬菌体肽库筛选与HIV-1p24抗原结合的多肽

通过噬菌体展示技术筛选出与HIV-1p24抗原结合的多肽,为用多肽辅助p24抗原检测提供实验基础.以重组p24抗原为靶蛋白,对噬菌体随机七肽库进行三轮筛选,用EUSA鉴定第三轮筛选到的噬菌体克隆与p24重组抗原的结合能力,再对噬菌体克隆进行测序分析,同时研究了ELISA中噬菌体加入量及多种封闭剂对噬菌体特异性结合能力的...     

A screening of phage displayed peptides for the recognition of fullerene (C60)

A novel approach to develop a peptide, that can recognize fullerene (C60) is described for affinity selection of phage displayed peptides from a combinatorial peptide library. Biopanning was performed using cyclic 7-mer peptide library against C60 films deposited on silicon (Si) substrate, and eluted phages with organic solvent. The phage, that recognized C60 films deposited on Si substrate, were obtained from biopanning. The nucleotides of the phage, coding a cyclic 7-mer peptide, were sequenced by standard method. Seventeen kinds of peptide displayed phages were obtained. One kind of peptide (peptide No. 4) displayed phage recognized the C60 films deposited on Si substrate. Peptide No. 4 displayed no affinity towards the Si substrate. The recognition event was monitored by a fluorescent immunoassay. Additionally, peptide No. 4 phage could recognize C60 in powder form, but not the graphite powder. This recognition event in powder form was also observed by a fluorescent immunoassay.     

Peptide presentation by bacteriophage P4

Abstract: This article focuses on bacteriophage P4 as a potential peptide display phage by exploring the possibility of using the P4 capsid decoration component, Psu, as a peptide carrier protein. Psu is non-essential for P4 growth but it enhances the stability of the P4 capsid by binding to its exterior. We have constructed a unique Sac I cloning site in the beginning of the psu gene. This site changes the third amino acid of Psu from Ser to Leu. This substitution does not destroy the binding of Psu to the P4 capsid. A synthetic oligonucleotide encoding a 10-amino acid peptide whose sequence is part of the human p62^c-myc protein, has been inserted into the Sac I site. The Psu_c-myc shows full capsid binding activity and reacts with monoclonal antibodies directed against the c-myc peptide. These results pave the way for the further development of a peptide display system based on bacteriophage p4.     

HWGMWSY, an unanticipated polystyrene binding peptide from random phage display libraries

Phage display is a powerful technique for the discovery of peptide ligands that bind to various targets; however, ambiguous results often appear. Peptide HWGMWSY has been isolated repeatedly in our laboratory and by other research groups dealing with different protein and nonprotein targets, which led to a hypothesis that it may be a target-unrelated peptide interacting with polystyrene plastic surfaces. We compared binding properties and amplification rate of phage clone displaying the peptide HWGMWSY, a previously confirmed plastic binding clone WHWRLPS, and a control phage clone ASVQERK. An enzyme-linked immunosorbent assay and a phage elution assay confirmed that phage clone HWGMWSY binds to polystyrene. Surface plasmon resonance measurements on the other hand excluded the possibility of binding to bovine serum albumin, a common blocking agent in phage display experiments. Amplification rates of the above-noted phage clones were not statistically different. We therefore conclude that phage clone HWGMWSY was isolated in different selection procedures as a result of its affinity to polystyrene.     

Phage display selection of peptides that inhibit metastasis ability of gastric cancer cells with high liver-metastatic potential

Organ-specific metastasis is an important character of cancer cells. Cancer cells that can metastasize to a special organ were thought to have different proteins in cell membrane, which might have potential utility as diagnostic markers and therapeutic targets. In the present work, based on high liver-metastatic gastric cancer cells, XGC9811-L, a screening approach with phage displayed peptide library, was successfully used to isolate 8-mer peptide ligands binding to the target cells. The phage20 had the highest binding efficiency to XGC9811-L cells, which also displayed remarkable cell specificity. Peptide20 that was displayed on phage20 could suppress the motility and invasion of XGC9811-L significantly. The adhesive ability of XGC9811-L to collagen IV was also inhibited by peptide20. Furthermore, phage20 could significantly reduce the incidence of liver metastasis of gastric cancer transplanted into nude mice and was also beneficial for the reduction the number of metastatic nodules in the liver. In conclusion, the phage display is an effective method to screen for the new molecules associated with organ-specific metastasis. The selected peptide20 can reverse the liver metastasis behavior of the gastric cancer cells.     

Selection and properties for the recognition of P19 embryonic carcinoma stem cells

The P19 cell is a pluripotent stem cell of murine teratocarcinoma. When treated with retinoic acid, P19 cells can be differentiated along a neural cell lineage in culture. To isolate peptides that bind to the stem cell, we employed a phage display technology with undifferentiated P19 cells as the target. To reduce nonspecific binding of phages to the cell surface, the phage libraries were preadsorbed to the differentiated P19 cells before each selection on the undifferentiated P19 cells. After eight rounds of the selection, No. 28 phage displaying ALPSTSSQMPQL-peptide was isolated. Immunofluorescence analysis revealed that No. 28 phage selectively binds to the undifferentiated P19 cells but not to the differentiated P19 cells or SHSY cell line. The chemically synthesized peptide ALPSTSSQMPQL presented on the No. 28 phage efficiently inhibited the binding of No. 28 phage to the undifferentiated P19 cells. This result confirmed that No. 28 phage binding to the cell was mediated by the displayed peptide. The identified peptide may be targeted to a marker expressed on the stem cell and thus become a practical tool for the isolation of somatic stem cells.     

Tumor specific phage particles promote tumor regression in a mouse melanoma model

Within cancer research, phage display libraries have been widely used for the identification of tumor targeting peptides and antibodies. Additionally, phages are known to be highly immunogenic; therefore we evaluated the immunotherapeutic potential of tumor specific phages to treat established solid tumors in a mouse model of melanoma. We developed two tumor specific phages, one derived from a peptide phage display library and one Fab expressing phage with known specificity, for the treatment of mice bearing palpable B16-F10 or B16/A2K^b tumors. Therapy in B16-F10 tumor bearing mice with tumor specific phages was superior to treatment with non-tumor specific phages and lead to delayed tumor growth and increased survival. In B16/A2K^b tumor bearing mice, therapy with tumor specific phages resulted in complete tumor regression and long-term survival in 50% of the mice. Histological analysis of tumors undergoing treatment with tumor specific phages revealed that phage administration induced a massive infiltration of polymorphonuclear neutrophils. Furthermore, phages induced secretion of IL-12 (p70) and IFN-γ as measured in mouse splenocyte culture supernatants. These results demonstrate a novel, immunotherapeutic cancer treatment showing that tumor specific phages can promote regression of established tumors by recruitment of inflammatory cells and induction of Th1 cytokines.     

Preferential selection of Cys‐constrained peptides from a random phage‐displayed library by anti‐glucitollysine antibodies

Phage‐displayed peptides recognized by two monoclonal antibodies against glucitollysine were selected. The most prominent feature of the peptide panel was the presence of paired Cys in most of them (21/24 peptides). The availability of a wide variety of peptides having differently spaced paired Cys, as well as truly linear Cys‐free peptides, gave the opportunity to explore the role of disulfide bridges in phage selection. Some Cys‐containing peptides came from a Cys‐flanked cyclic 9‐mer library, but most of them (18/21) were derived from a totally random 12‐mer library, and hence the presence of Cys was dictated by the selector antibodies. Motifs shared by several peptides (potentially involved in binding) often contained or were flanked by Cys residues. Binding of all Cys‐containing phage‐displayed peptides was abolished/decreased after a reducing treatment. Screening a random peptide library (without invariant Cys residues) is powerful enough to clearly reveal the need, preferences, and diversity of Cys‐mediated structural constraints for recognition. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Screening and identification of a specific peptide binding to hepatocellular carcinoma cells from a phage display peptide library

To screen and identify the novel probe markers binding hepatocellular carcinoma specifically and sensitively, a phage‐displayed 12‐mer peptide library was used to make biopanning with the modified protocols on HepG2 cells. After four rounds of panning, the consensus sequences were obtained, and the PC28, a phage clone with most specific and sensitive binding to HepG2 cells, was identified as the best positive clone. The peptide probe HCSP4 (sequence SLDSTHTHAPWP) was synthesized based on the sequencing result of PC28. The specificity and sensitivity of HCSP4 were primarily analyzed using immunofluorescence, flow cytometry, and other methods. The results show that HCSP4 can bind to hepatocellular carcinoma cells with satisfactory specificity and sensitivity. It may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

A novel peptide specifically targeting ovarian cancer identified by in vivo phage display

Discovery of peptide ligands that can target human ovarian cancer and deliver chemotherapeutics offers new opportunity for cancer therapy. The advent of phage‐displayed peptide library facilitated the screening of such peptides. In vivo screening that set in a microanatomic and functional context was applied in our study, and a novel peptide WSGPGVWGASVK targeting ovarian cancer was isolated. The phage clone PC3‐1 displaying peptide WSGPGVWGASVK can gain effective access to accumulate in the tumor sites after intravenous injection while reducing its accumulation in normal organs. Positive immunostaining of PC3‐1 was located in both sites of tumor cells and tumor blood vessels, which resulted in a diffuse binding pattern through the tumor. In vitro study results confirmed the capability of peptide WSGPGVWGASVK binding to and being internalized by both tumor cells and angiogenic endothelial cells. Flow cytometry analysis revealed that the peptide bound to SKOV3 cells with Kd value of 5.43 ± 0.4 μM. Taken together, it suggested that peptide WSGPGVWGASVK is a lead candidate for delivering therapeutics to penetrate into tumors. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography

Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by dis-playing it on the surface of filamentous bacteriophage fd. Phosphorylation in vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-mer peptide library phosphorylated by cAPK was selected with ferric (Fe3 ) chelalion affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Their in vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typ     

A consensus tetrapeptide selected by phage display adopts the conformation of a dominant discontinuous epitope of a monoclonal anti-VWF antibody that inhibits the von Willebrand factor-collagen interaction

Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg963, Pro981, Asp1009, Arg1016, Ser1020, Met1022, and His1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser1020, Arg1016, Pro981, and Trp982 in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser1020 and Arg1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp982. In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.     

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog,Odorrana livida

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana‐BI. The cDNA sequencing was obtained by ‘shotgun’ cloning. The amino acid sequence of the mature peptide was identified as Gly‐Leu‐Leu‐Ser‐Gly‐Lys‐Ser‐Val‐Lys‐Gly‐Ser‐Ile‐OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI‐TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana‐BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana‐BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

A novel CDKN2A variant (p16L117P) in a patient with familial and multiple primary melanomas

Germline mutations in CDKN2A (p16) are commonly found in patients with family history of melanoma or personal history of multiple primary melanomas. The p16 tumor suppressor gene regulates cell cycle progression and senescence through binding of cyclin‐dependent kinases (CDK) and also regulates cellular oxidative stress independently of cell cycle control. We identified a germline missense (c.350T>C, p.Leu117Pro) CDKN2A mutation in a patient who had history of four primary melanomas, numerous nevi, and self‐reported family history of melanoma. This particular CDKN2A mutation has not been previously reported in prior large studies of melanoma kindreds or patients with multiple primary melanomas. Compared with wild‐type p16, the p16^L117P mutant largely retained binding capacity for CDK4 and CDK6 but exhibited impaired capacity for repressing cell cycle progression and inducing senescence, while retaining its ability to reduce mitochondrial reactive oxygen species. Structural modeling predicted that the Leu117Pro mutation disrupts a putative adenosine monophosphate (AMP) binding pocket involving residue 117 in the fourth ankyrin domain. Identification of this new likely pathogenic variant extends our understanding of CDKN2A in melanoma susceptibility and implicates AMP as a potential regulator of p16.     

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.     

Phage-displayed peptide targeting on the Puumala hantavirus neutralization site.

We have selected ligands for Puumala hantavirus, the causative agent of nephropathia epidemica, from a seven-amino-acid peptide library flanked by cysteines and displayed on a filamentous phage. To direct the selection to areas on the virus particle which are essential for infection, phages were competitively eluted with neutralizing monoclonal antibodies specific for the viral glycoproteins. The selected phage populations were specific for the same sites as the antibodies and mimicked their functions. The peptide insert, CHWMFSPWC, when displayed on the phages, completely inhibited Puumala virus infection in cell culture at the same effective concentration as the eluting antibody specific for envelope glycoprotein G2. The binding of the phage clones to the virus and inhibition of infection were not necessarily coincident; Pro-6 was critical for virus inhibition, while consensus residues Trp-2 and Phe-4 were essential for binding. The strategy described can be applied to any virus for production of molecules mimicking the effect of neutralizing antibodies.