Development of a tactical screening method to investigate the characteristics of functional peptides

Using spot-synthesized peptide arrays, a functional peptide can be screened as a high-binding peptide for a target molecule. We have developed a rational screening method for functional peptides by analyzing the physicochemical rules of high-binding peptide sequences. To screen the peptides simply and strategically, we prepared an exhaustive 4-mer peptide library consisting of 256 peptides (4⁴ = 256) characterized by four physicochemical groups of 20 amino acids: Group 1, non-charged hydrophobic amino acids; Group 2, non-charged hydrophilic amino acids; Group 3, positive-charged hydrophilic amino acids; Group 4, negative-charged hydrophilic amino acids. First, our previous screening data from cell adhesion, bile acid-binding, and nanoparticle-binding peptides were applied to the four-category analysis, and target-specific physicochemical characteristics were obtained. We then prepared an exhaustive 4-mer peptide library using these four physicochemical groups, and screened for high-binding peptides that bind model proteins interleukin-2 and IgG. We obtained individual physicochemical rules for high-binding peptides: group 1 or 4 amino acids in position (P) 1, group 1 in P2 and P4 for IL-2, and group 2 and 3 amino acids at all position for IgG. Therefore, this system, which employs the use of a simple and strategic peptide library, will be useful in the development of functional peptides.     

Functional characterization of CTL against gp100 altered peptide ligands

In this study, four modified gp100 peptides were designed by combining amino acids from the melanoma peptide antigen gp100((209-217)) with preferred primary and auxiliary HLA-A *0201 anchor residues previously identified from combinatorial peptide library screening with recombinant HLA-A*0201. These modified peptides demonstrated stronger binding affinity for the HLA-A*0201 molecule compared to wild-type gp100 peptide. Nine CTL lines generated from patients immunized with the g209-2 M peptide and one CTL line from a non-immunized patient were tested for the ability to respond to these modified gp100 peptides. Stimulation of CTL by two of four modified peptides induced higher levels of IFN-gamma secretion than the wild-type gp100 peptide, demonstrating that higher peptide binding affinity for HLA molecules does not necessarily equate to functional activity of CTL. Two major and one minor CTL recognition pattern were observed, irrespective of previous peptide immunization, suggesting that multiple, rationally designed modified tumor peptides for the same epitope stimulate a broad CTL response by activating multiple CTL capable of cross-reacting with the natural antigenic peptide.     

Peptides derived from a secretory yeast library restore factor VIII activity in the presence of an inhibitory antibody

The development of autoantibodies against factor VIII represents one of the major complications in the treatment of hemophilia A patients. We have employed a novel library system to obtain peptides that specifically neutralize the interaction between factor VIII and these inhibitors. The random peptides are presented as carboxy-terminal extensions of the eukaryotic initiation factor 5a, an intracellular protein with a molecular mass of 18 kDa. These random peptides formed an unique binding site, as demonstrated by molecular simulations using the computer programs InsightII and GROMACS. The library was screened to identify peptides binding to the murine monoclonal anti-factor VIII antibody ESH8 and to inhibitors derived from patients with factor VIII antibodies. Ten peptides binding to ESH8 were identified. Their specificity was confirmed by displacement assays. Two peptides with the sequences STKTLGRPLHGPAGPVEGGALAGVAEDADLVTAVSGR and YHCKREDLTDRDATCALRQPPQAVRGLGPRVTAVSGR showed the ability to restore the factor VIII activity from 33% up to approximately 90% in functional tests performed in vitro. Three candidates for binding to factor VIII antibodies derived from four different patient's sera were achieved. Three fusion proteins with the peptide sequences PQLGSRRSTTPSLTFQNASWFPAGGPCARSNRG, SGSRQVCKLARSLQPF and WERGRRVGAQVRHARHLVARVLDGAGHQARLTAVNGP bound to inhibitors derived from different patients. Furthermore, two of the obtained fusion proteins with the peptide sequences RHWTALGPAPTHTCADLNYPLLS and WERGRRVGAQVRHARHLVARVLDGAGHQARLTAVNGP did also bind to the monoclonal antibody ESH8. This study demonstrates the potential of this system to identify peptides that inhibit the activity of potent inhibitory antibodies and also shows potential as a method for screening of bioactive peptides.     

A serotype-specific epitope of dengue virus 1 identified by phage displayed random peptide library

Abstract From a panel of monoclonal antibodies of dengue viruses, a serotype-specific epitope of dengue virus 1 was screened from a random peptide library displayed on phage. The epitope was the determinant reactive with monoclonal antibody 15F3-1 that was specific to dengue 1. The screening was monitored by a dot blotting procedure, and after three rounds of screening a consensus motif, HRYSWK, was found. This sequence matches the sequence HKYSWK, corresponding to the amino acid residues 885–890 of polyprotein or residues 111–116 of the non-structural protein 1 of dengue virus serotype 1. The linear epitope was confirmed by testing the antigenicity of chemically synthesized 8-branched peptide.     

Functional screening of a retroviral peptide library for MHC class I presentation

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.     

A new hybrid resin for stepwise screening of peptide libraries combined with single bead Edman sequencing

A library system was developed for the discovery of bioactive peptides. Library synthesis and peptide sequencing was performed on a solid support while the screening for bioactivity was done with peptides in solution. The peptides were synthesized by split and mix, one-bead–one-peptide library synthesis, using a Tentagel S-NH₂ solid support with a loading of approximately 100 pmol/bead. The major part of the peptide was connected to the support by a single acid-labile linker and a minor part of the peptide was acid-stabile attached to the polymer. The percentage of acid-stabile attached peptides could easily be controlled during modification of the amino functionalities of the resin at the start of the process. The cleavage rate of the acid-labile attached peptide from the resin depends on the composition of the cleavage mixture. When cleavage conditions were carefully controlled, a three-step partial cleavage protocol allowed for convergent bioactivity screening on peptide libraries using only one type of acid-labile linker. The partial cleavage and convergent screening procedure was repeated three times, after which the bead containing the bioactive peptide was sequenced. As such a bead still contained acid-stabile attached peptide, the Edman sequencing was straightforward and repetitive yields were excellent because the immobilized peptide was not washed out. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

直接用噬菌体肽进行功能性分析的研究

通过对胰蛋白酶抑制剂的筛选发现,噬菌体肽库不仅可以用于亲和性筛选,而且噬菌体肽（ｐｈａｇｅｐｅｐｔｉｄｅ——ｐｅｐｔｉｄｅｄｉｓｐｌａｙｉｎｇｏｎｐｈａｇｅ）可以直接用于对亲和筛选得到的克隆进行胰蛋白酶抑制实验,即所谓的功能性筛选．功能性筛选前后的测序结果表明用噬菌体肽直接进行功能性筛选确实能够筛去大部分的非抑制剂克隆．     

One-bead, one-compound peptide library sequencing via high-pressure ammonia cleavage coupled to nanomanipulation/nanoelectrospray ionization mass spectrometry

Biological screening of one-bead, one-compound (OBOC) combinatorial peptide libraries is routinely carried out with the peptide remaining bound to the resin bead during screening. After a hit is identified, the bead is isolated, the peptide is cleaved from the bead, and its sequence is determined. We have developed a new technique for cleavage of peptides from resin beads whereby exposure of a 4-hydroxymethyl benzoic acid (HMBA)-linked peptide to high-pressure ammonia gas led to efficient cleavage in as little as 5 min. Here we also report a new method of extracting peptide from individual library beads for its introduction into a mass spectrometer that uses nanomanipulation combined with nanoelectrospray ionization mass spectrometry (NSI MS). Single beads analyzed by nanomanipulation/NSI MS were found to give identical MS results to those of bulk samples. Detection of 18 unique cleaved peptides 1 to 8 amino acids in length, and sequencing of 14 different peptide sequences 4 to 8 amino acids in length, was demonstrated on a combination of bulk samples and ones from individual beads of an OBOC library. The method was highly reproducible, with 100% of attempts to extract peptide resulting in high-quality MS data. This new collection of techniques allows rapid, reliable, environmentally responsible sequencing of hit beads from combinatorial peptide libraries.     

Preparation, analysis and antibody binding studies of simultaneously synthesized soluble and cellulose-bound HIV-1 p24 peptide epitope libraries

Summary Epitope libraries of the HIV-1 p24 epitope GATPQDLNTM, recognized by the murine monoclonal antibody CB 4-1, were prepared by simultaneous synthesis on single resin supports (solution phase library) and on a continuous cellulose membrane support (solid phase-bound library) Each position of the epitope was replaced by 19 l-amino acids (cysteine omitted) in the soluble library or by 20 l-amino acids in the cellulose-bound library. The soluble library was synthesized by simultaneously incorporating equimolar amino acid mixtures at each position of the epitope or by synthesizing single epitope analogues. The peptide mixtures were subsequently analyzed by HPLC, CZE and MALDI-TOF mass spectrometry. Double coupling of equimolar amino acid mixtures of either 0.8 equiv (coupling at epitope positions 6–10) or 1.5 equiv (coupling at epitope positions 1–5) resulted in approximately equimolar incorporation of all single components of the mixture. The mixtures were then separated by preparative HPLC, and the peptides or peptide mixtures of single fractions were isolated and analyzed for binding CB 4-1. The results were compared with those obtained from antibody binding studies using the cellulose-bound epitope library. The affinity constants of the soluble peptide variants qualitatively correlated with the binding of CB 4-1 to single cellulose-bound analogues. Both approaches allowed the rapid identification of key residues in antibody binding, thus giving insight into the molecular nature of this antibody-peptide interaction.     

脂多糖保守表位模拟肽的筛选与鉴定

用针对脂多糖保守表位的单抗2B4对噬菌体随机12肽库进行亲和筛选,通过噬菌体ELISA实验及脂多糖(LPS)竞争抑制实验鉴定阳性克隆.经三轮筛选后,与抗体结合的噬菌体得到明显富集,噬菌体ELISA结果显示,阳性率达80%.将其中12个阳性噬菌体克隆做鼠伤寒杆菌和大肠杆菌LPS竞争抑制实验,抑制作用非常明显,有良好的剂量依赖关系,证明这12个克隆与LPS具相似表位.DNA测序并推导噬菌体展示肽的氨基酸序列为,GPPQWFFSQPQL（5/12,41.7%）,LPQYFWNTATTA（3/12,25%）,FPQNHWNVPWAT（2/12,16.6%）,HSQSFWNAPLAM和AHPWTHGYFPPL（1/12,8.3%）.实验结果表明,用2B4抗体筛选到的噬菌体短肽克隆可模拟保守表位,即脂多糖的模拟肽（位）.     

A One-Bead One-Peptide Combinatorial Library Method for B-Cell Epitope Mapping

The one-bead one-peptide combinatorial library method represents a powerful approach to the discovery of binding peptides for various macromolecular targets. It involves the synthesis of millions of peptides on beads such that each bead displays only one peptide entity. The peptide–beads that interact with a specific macromolecular target are then isolated for structure determination. We have applied this method to discovering peptide ligands for several murine monoclonal antibodies: (i) anti-β-endorphin (continuous epitope), (ii) anti-vmos peptide, (iii) anti-human insulin (discontinuous epitope), and (iv) surface immunoglobulins (μκ) of two murine B-cell lymphoma cell lines (antigen unknown).     

Epitope identification by polyclonal antibody from phage-displayed random peptide library

Screening of bioactive peptides from random peptide libraries using monoclonal antibodies as ligates is an effective method to define epitopes of protein antigens. However, it is thought that polyclonal antibodies might also serve as promising ligates for screening. We illustrate this approach by using recombinant human lymphotoxin (rhLT) polyclonal antibody as a model. The procedure consists in (a) affinity purification of polyclonal antibody to obtain the monospecific antibody, (b) screening against a phage-displayed random peptide library using the affinity-purified antibody, (c) plating the enriched phage on agar plates, randomly picking clones, and selecting the positive ones by dot blotting, (d) DNA sequencing of the positive clones and conducting a homology search against the protein sequence databank, and (e) confirming the epitopes by chemical peptide synthesis. By employing this procedure, we identified a dominant epitope RQHPKM, located at residues 15–20 of the human lymphotoxin amino acid sequence. The usefulness of this general procedure is discussed.     

应用噬菌体展示随机肽库淘筛mAb 5H5识别的抗原表位

本文利用噬菌体随机9肽库探索汉滩病毒（HTNV）核衣壳蛋白（NP）B细胞抗原表位。以抗HTNV NP单克隆抗体(mAb）5H5作为筛选分子,生物淘洗噬菌体递呈的随机9肽库。阳性克隆经夹心ELISA、竞争ELISA鉴定后,随机挑取10个克隆,DNA测序,与HTNV76－118株S基因进行同源性分析。结果显示筛选到的噬菌体能特异地与5H5结合,这种结合可被天然抗原所抑制。10个克隆的氨基酸序列相同,均为VRDAEEQYE,与76－118株NP氨基端的aa25-33一致。证实了该线性表位是mAb 5H5识别的表位,噬菌体肽库有助于病毒抗原表位的确定。     

Phage display: Concept,innovations, applications and future

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.     

Identification of a ligand for IgG-Fc derived from a soluble peptide library based on fusion proteins secreted by S. cerevisiae

Biological libraries are important tools in the development of new peptide-based compounds. Here, we describe the use of a soluble peptide library system as a complementary tool in the field of ligand development. Random peptides were expressed in S. cerevisiae as carboxy-terminal extensions of the eukaryotic initiation factor 5a (eIF5a) and secreted into the culture supernatant. Expression and screening of this library were performed in a microwell format. As an example of this versatile approach, we describe the identification of a ligand for the human IgG-Fc fragment. Ligands binding IgG-Fc show great potential in a wide variety of applications including development of therapeutics, streamlining the large-scale purification of antibodies, and applications in diagnostic tests. We demonstrated the utility of this system. After screening only 6160 clones, we identified a ligand with the peptide sequence of TRRRTCSPPTWPRARARSTPSGCSSTGPSANRG. An affinity constant of 3.9 x 10(5) M(-1) was determined by a biosensor method. Handling and maintenance of this library is conceptually simple and highly applicable for automated high-throughput systems.     

A simple and efficient maleimide-based approach for peptide extension with a cysteine-containing peptide phage library

Although peptide-based molecules are known to have therapeutic potential, the generation of phage focused libraries to optimize peptides is effort-consuming. A chemical method is developed to extend a maleimide-conjugated peptide with a cysteine-containing random-peptide phage display library. As a proof of concept, a 15-mer epidermal growth factor receptor (EGFR)-binding peptide was synthesized with a maleimide group at its C-terminus and then conjugated to the cysteine-containing library. After panning and screening, several extended peptides were discovered and tested to have a higher affinity to EGFR. This strategy can have broad utility to optimize pharmacophores of any modalities (peptides, unnatural peptides, drug conjugates) capable of bearing a maleimide group     

HIV蛋白质优势B细胞抗原表位的筛选、重组表达与抗原性鉴定

设计了一种新的病原体蛋白质B细胞抗原表位的筛选和重 组表达方法。不须使用抗原,而通过交替用病人血清IgG抗体“淘洗”(biopanning)随机肽库和用正常人血清IgG反向吸附,来获得特异抗原表位资料。用HIV病人血清的IgG抗体淘洗噬菌体递呈随机十二肽库,再以正常人IgG抗体吸附,筛选到了能和HIV病人血清发生特异反应的噬菌体克隆,经ELISA、DNA测序等,成功地筛选出了位于HIV gp41外膜蛋白、高亲和力、构型特异的优势B细胞抗原表位(602GCSGKLICTTNV613)。用大肠杆菌硫氧还蛋白作为骨架,在其活性部位以“内融合”形式重组表达了该抗原表位,纯化的重组蛋白具有良好的抗原性,能与HIV(+)IgG抗体及艾滋病人血清呈特异反应,表明本技术路线可以有 效地进行HIV蛋白质的B细胞抗原表位筛选和重组表达。此方法也可移植于其它病原微生物抗原或自身抗原的表位研究,继而为基于抗原表位水平的特异诊断试剂的研制、疫苗的设计提供依据。     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

应用噬菌体随机肽库技术筛选丙肝病毒NS3抗原模拟表位

以抗 HCVNS3的单克隆抗体作为固相筛选分子 ,对人工合成的噬菌体随机 12肽库进行 5轮“吸附 洗脱 扩增”的筛选过程 ,随机挑取 4 2个克隆 ,经噬菌体酶联免疫吸附法 (ELISA)鉴定并进行交叉反应实验以及竞争抑制性结合实验 ,最后对所选克隆进行DNA序列分析 ,以确定HCVNS3抗原的模拟表位。经噬菌体富集后 ,从随机筛选的 4 2个克隆中得到 11个阳性克隆 ,确定氨基酸序列XXIXXXXMSNXX为HCVNS3的模拟表位。我们用噬菌体12肽库成功筛选得到HCVNS3的模拟表位 ,为开展用HCV模拟表位探索HCV的防治研究创造了条件