Phagemid encoded small molecules for high throughput screening of chemical libraries

A new strategy for monovalently displaying small molecules on phage surfaces was developed and applied to high throughput screening for molecules with high binding affinity to the target protein. Peptidyl carrier protein (PCP) excised from nonribosomal peptide synthetase was monovalently displayed on the surface of M13 phage as pIII fusion proteins. Small molecules of diverse structures were conjugated to coenzyme A (CoA) and then covalently attached to the phage displayed PCP by Sfp phosphopantetheinyl transferase. Because Sfp is broadly promiscuous for the transfer of small molecule linked phosphopantetheinyl moieties to apo PCP domains, this approach will enable displaying libraries of small molecules on phage surfaces. Unique 20-base-pair (bp) DNA sequences were also incorporated into the phagemid DNA so that each compound displayed on the phage surface was encoded by a DNA bar code encapsulated inside the phage coat protein. Single round selection of phage displayed small molecules achieved more than 2000-fold enrichment of small molecules with nM binding affinity to the target protein. The selection process is further accelerated by the use of DNA decoding arrays for identifying the selected small molecules.     

Identification of calpain substrates by ORF phage display

Substrate identification is the key to defining molecular pathways or cellular processes regulated by proteases. Although phage display with random peptide libraries has been used to analyze substrate specificity of proteases, it is difficult to deduce endogenous substrates from mapped peptide motifs. Phage display with conventional cDNA libraries identifies high percentage of non-open reading frame (non-ORF) clones, which encode short unnatural peptides, owing to uncontrollable reading frames of cellular proteins. We recently developed ORF phage display to identify endogenous proteins with specific binding or functional activity with minimal reading frame problem. Here we used calpain 2 as a protease to demonstrate that ORF phage display is capable of identifying endogenous substrates and showed its advantage to re-verify and characterize the identified substrates without requiring pure substrate proteins. An ORF phage display cDNA library with C-terminal biotin was bound to immobilized streptavidin and released by cleavage with calpain 2. After three rounds of phage selection, eleven substrates were identified, including calpastatin of endogenous calpain inhibitor. These results suggest that ORF phage display is a valuable technology to identify endogenous substrates for proteases.     

利用pⅧ展示系统改进噬菌体抗体芯片

将展示单链抗体的重组噬菌体与羧基终止的硅片偶联, 制成噬菌体抗体芯片, 可用于检测多类蛋白质和蛋白质组. 通常抗体被展示于噬菌体外壳蛋白pⅢ上, 由此制备的芯片灵敏度和信噪比较低. 我们选用凝血酶特异的单链抗体为代表, 比较了pⅢ展示系统和pⅧ展示系统制成芯片的检测效果. 由于pⅧ展示系统的融合蛋白拷贝数多, 所受空间位阻小, 大幅度提高了噬菌体抗体芯片的灵敏度和信噪比, 有望用于制备新型蛋白质芯片.     

Design and screening of M13 phage display cDNA libraries

The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF) libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS) will be presented.     

Material binding peptides for nanotechnology

Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.     

Screening for protease substrate by polyvalent phage display

Proteases are key regulators of many physiological and pathological processes [1,2], and are recognized as important and tractable drug candidates. Consequently, knowledge of protease substrate recognition and specificity promotes identification of biologically relevant substrates, helps elucidating a protease's biological function, and the design of specific inhibitors. Traditional methods for establishing substrate recognition profiles involve the identification of the scissile bond within a given protein substrate by proteomic methods such as Edman degradation. Then, synthetic peptide variants of this sequence can be screened in an iterative fashion to arrive at more optimized substrates. Even though it can be fruitful, this iterative strategy is biased toward the original substrate sequence and it is also tremendously cumbersome. Furthermore, it is not amenable to high throughput analysis. In 1993, Matthew & Wells presented a method for the use of monovalent "substrate phage" libraries for discovering peptide substrates for proteases, in which more than 10(7) potential substrates can be tested concurrently [3]. A library of fusion proteins was constructed containing randomized substrate sequences placed between a binding domain and the gene III coat protein of the filamentous phage, M13, which displays the fusion protein and packages the gene coding for it inside. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). This method allows one to rapidly survey the substrate recognition and specificity of individual or closely related members of proteases. Over the past decade, substrate phage screening has shown terrific utility in rapidly determining protease specificity and characterization of substrate recognition profile of proteases. In some cases, the structural insights of the catalytic domain were obtained from comparison of substrate specificity among closely related family of proteases [4-6]. The number of proteases (from various classes) characterized by this approach testifies to its power. Since the initial development of substrate phage library, different versions of the substrate phage cloning vectors have been constructed to further improve the utility of substrate phage display. This review will provide an overview of the construction of substrate phage display libraries, screening of substrate phage libraries, examples of application, summary and future directions.     

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins

BACKGROUND: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor kappa B (NF-kappa B) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. RESULTS: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-kappa B p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-kappa B p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:10(8) and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. CONCLUSIONS: A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.     

Display cloning: functional identification of natural product receptors using cDNA-phage display.

BACKGROUND: The identification of cellular targets has traditionally been the starting point for natural product mode of action studies and has led to the understanding of many biological processes. Conventional experimental approaches have depended on cell-based screening and/or affinity chromatography. Although both of these techniques aid in the discovery of protein cellular targets, a method that couples protein identification with gene isolation would be extremely valuable. RESULTS: A procedure for the direct cloning of cellular proteins, based on their affinity for natural products, using cDNA phage display has been developed. The technique is referred to as display cloning because it involves the cloning of proteins displayed on the surface of a bacteriophage particle. The approach has been established by isolating a full-length gene clone of FKBP12 (FK506-binding protein) from a human brain cDNA library using a biotinylated FK506 probe molecule. During the affinity selection, the FKBP12 gene emerged as the dominant library member and was the only sequence identified after the second round of selection. CONCLUSIONS: The development of display cloning greatly facilitates the investigation of ligand-receptor interaction biology and natural product mode of action studies. This procedure utilizes heterologous protein display on infectious phage, which allows the amplification and repeated selection of putative sequences, leading to unambiguous target identification. In addition, the direct connection of a functional protein to its gene sequence eliminates the subsequent cloning step required with tissue homogenate or cell lysate affinity methods, allowing direct isolation of an expressible gene sequence.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

The baculovirus expression system as a tool for generating diversity by viral surface display

It has become a major goal of molecular biologists, biochemists, and immunologists to be able to modulate the structure of proteins, in order to increase their antigenicity, alter their biological properties and/or explore their function. Based on the concept of bacterial phage display, by which proteins are being selected and analyzed in conjunction with their genetic information, eukaryotic systems have been investigated for their use in generating biomolecular diversity. The advantage of posttranslational modification and the possible harbouring of structural complex proteins has lead scientists to include eukaryotic systems in the wide field of molecular design. The ideal expression vectors for surface display are eukaryotic viruses, that allow large gene insertions, efficiently present foreign proteins on the particle surface, are easy to propagate and, if possible, not pathogenic to humans. By inserting peptides into a native virus coat protein or by expressing foreign proteins as coat protein fusion proteins or linked to specific anchor domains it becomes possible to display polypeptides of interest on the surface of replicating particles. A variety of different strategies are currently under investigation in order to utilize the baculovirus insect cell expression system for efficient display on the surface of virus particles as well as on the surface of virally infected insect cells. Increasing the transfection efficiency, optimizing cloning procedures, and establishing applicable selection methods have lead to the development of a powerful tool for drug screening and ligand screening.     

Filamentous bacteriophage stability in non-aqueous media.

BACKGROUND: Filamentous bacteriophage are used as general cloning vectors as well as phage display vectors in order to study ligand-receptor interactions. Exposure to biphasic chloroform-water interface leads to specific contraction of phage, to non-infective I- or S-forms. RESULTS: Upon exposure, phage were inactivated (non-infective) at methanol, ethanol and 1-propanol concentrations inversely dependent upon alcohol hydrophobicity. Infectivity loss of phage at certain concentrations of 1-propanol or ethanol coincided with changes in the spectral properties of the f1 virion in ultraviolet fluorescence and circular dichroism studies. CONCLUSIONS: The alcohols inactivate filamentous phage by a general mechanism--solvation of coat protein--thereby disrupting the capsid in a manner quite different from the previously reported I- and S-forms. The infectivity retention of phagemid pG8H6 in 99% acetonitrile and the relatively high general solvent resistance of the phage strains studied here open up the possibility of employing phage display in non-aqueous media.     

A phage display system with unnatural amino acids

We report a general method to display peptide-containing unnatural amino acids on filamentous M13 phage. Five distinct unnatural amino acids were site-specifically incorporated at the N-terminal of the M13 phage minor coat protein pIII. Phages that contain p-azidophenylalanine can undergo a highly specific azide-alkyne [3 + 2] cycloaddition reaction with an alkyne-derivatized fluorophore. The generalization of phage display to include unnatural amino acids should significantly increase the scope of phage display technology.     

Isolation of a Potential Anchoring Motif Based on Proteome Analysis of Escherichia coli and Its Use for Cell Surface Display

For bacterial cell surface display, the target protein needs to be linked to an anchoring motif, and it is essential to choose an appropriate anchoring motif for efficient and stable display of the protein on the cell surface. To isolate a potential anchoring motif that would allow a stable and enhanced display of target proteins on the surface of an Escherichia coli host, we analyzed the outer membrane proteome of E. coli. On the basis of this proteomic analysis, the outer membrane protein X (OmpX), which has a small, monomeric β-barrel structure and is highly expressed, was selected as a potential anchoring motif. The role of OmpX as an anchoring motif for cell surface display was demonstrated using three important industrial enzymes: endoxylanase, lipase, and alkaline phosphatase. Two different positions (Lys¹²², Val¹⁶⁰) in the extracellular loops of OmpX were examined for C-terminal fusion, and the biological activities and localization of the displayed enzymes were analyzed. All three enzymes examined were efficiently displayed on the E. coli cell surface with high activity. These results reveal that the use of OmpX as an anchoring motif is an efficient method to display functional enzymes on the surface of an E. coli host.     

Phage display: selecting straws instead of a needle from a haystack

An increasing number of peptides with specific binding affinity to various protein and even non-protein targets are being discovered from phage display libraries. The power of this method lies in its ability to efficiently and rapidly identify ligands with a desired target property from a large population of phage clones displaying diverse surface peptides. However, the search for the needle in the haystack does not always end successfully. False positive results may appear. Thus instead of specific binders phage with no actual affinity toward the target are recovered due to their propagation advantages or binding to other components of the screening system, such as the solid phase, capturing reagents, contaminants in the target sample or blocking agents, rather than the target. Biopanning experiments on different targets performed in our laboratory revealed some previously identified and many new target-unrelated peptide sequences, which have already been frequently described and published, but not yet recognized as target-unrelated. Distinguishing true binders from false positives is an important step toward phage display selections of greater integrity. This article thoroughly reviews and discusses already identified and new target-unrelated peptides and suggests strategies to avoid their isolation.     

Targeting of phage display vectors to mammalian cells

Phage display libraries offer a strategy to isolate peptide ligands to target proteins and to define potential interaction sites between proteins. Recent studies have indicated a novel utility for phage display in that bacteriophage engineered to express peptide ligands to specific cell surface receptors are internalized by mammalian cells. Thus, reporter genes such as green fluorescent protein and lacZ harbored in the phage genome can be delivered to mammalian cells using targeting peptides displayed on the surface of phage. There is also the possibility to generate novel types of peptide libraries expressed intracellularly using a phage capable of inducing expression of its coding genes in human cells.     

Accelerated screening of phage-display output with alkaline phosphatase fusions

When using multiple targets and libraries, selection of affinity reagents from phage-displayed libraries is a relatively time-consuming process. Herein, we describe an automation-amenable approach to accelerate the process by using alkaline phosphatase (AP) fusion proteins in place of the phage ELISA screening and subsequent confirmation steps with purified protein. After two or three rounds of affinity selection, the open reading frames that encode the affinity selected molecules (i.e., antibody fragments, engineered scaffold proteins, combinatorial peptides) are amplified from the phage or phagemid DNA molecules by PCR and cloned en masse by a Ligation Independent Cloning (LIC) method into a plasmid encoding a highly active variant of E. coli AP. This time-saving process identifies affinity reagents that work out of context of the phage and that can be used in various downstream enzyme linked binding assays. The utility of this approach was demonstrated by analyzing single-chain antibodies (scFvs), engineered fibronectin type III domains (FN3), and combinatorial peptides that were selected for binding to the Epsin N-terminal Homology (ENTH) domain of epsin 1, the c-Src SH3 domain, and the appendage domain of the gamma subunit of the clathrin adaptor complex, AP-1, respectively.     

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.     

Expression and Characterization of a Recombinant Truncated Capsid Protein of Hepatitis E Virus in Pichiapastoris

Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus(HEV).HEV is a nonenveloped virus that has been classified in the family of Caliciviridae.The virus appears to be a polyadenylated,positive-stranded RNA virus with three major open reading frames(ORFs).The capsid protein of HEV is encoded by the open reading frame 2(ORF2).We attempted to produce a truncated capsid protein,designed p293,in Pichia pastoris.The p293 gene encoding amino acids(aa) 382-674 of HEV ORF2 was designed based on the full length of HEV ORF2,cloned into the yeast vector pPIC9K,and expressed in P.pastoris strain GS115.SDS-PAGE and Western blotting demonstrated that the recombinant protein p293 could well be expressed in P.pastoris.Under optimized conditions (culture medium pH,6.0―6.5;methanol concentration added daily,3.0%;inoculum density,OD600=60;induction time point,72―96h),the yield of soluble p293 was approximately 80 mg/L.We also observed p293 secretory expressed in P.pastoris to be 30 nm viral like particles by using electron microscopy.These results show that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV,and serve as a useful antigen for both diagnostic and vaccine purposes.