Phage display in pharmaceutical biotechnology

Over the past year, methods for the construction of M13 phage-display libraries have been significantly improved and new display formats have been developed. Phage-displayed peptide libraries have been used to isolate specific ligands for numerous protein targets. New phage antibody libraries have further expanded the practical applications of the technology and phage cDNA libraries have proven useful in defining natural binding interactions. In addition, phage-display methods have been developed for the rapid determination of binding energetics at protein-protein interfaces.     

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.     

Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

Phage display of antibody fragments from natural or synthetic antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and sc-dsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv.     

Exploring and designing protein function with restricted diversity

Combinatorial libraries with restricted diversity can be used to rapidly map binding energetics across protein interfaces. Shotgun scanning strategies have been used for alanine scanning and for alternative mutagenesis schemes that provide high-resolution functional views of binding interfaces. In addition, synthetic antibodies have been derived from na?ve libraries restricted to a binary code to explore the minimal requirements for molecular recognition. These studies shed light on the underlying principles governing molecular recognition, and provide rapid yet quantitative alternatives to conventional biophysical methods for exploring protein structure and function.     

Engineering M13 for phage display

Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display. These improvements expand the utility of phage display as a powerful tool in modern biotechnology.     

The basic structure of filamentous phage and its use in the display of combinatorial peptide libraries

Combinatorial peptide libraries have been playing a major role in the search for new drugs, ligands, enzyme substrates, and other specifically interacting molecules. The principal features of these libraries require a versatile repertoire, an easily identifiable tag for each of the library members, a simple method of synthesis, and a compability with the biochemical milieu. Two types of combinatorial libraries are in use: synthetic libraries and biological (mainly phage display) ones. An advantage of the biological libraries is due to the ability of each of the library members to replicate itself and to the fact that they carry their own coding sequences. The uniqueness of filamentous phage is that of its five virion proteins, three can tolerate the insertion of foreign peptides, each in a distinctive manner. The major coat protein, pVIII, is capable of displaying hundreds of peptide copies over the phage virion, pIII can display either one or five copies, and pVI, as opposed to the first two, displays its peptides such that the carboxy terminus is oriented outward. A major drawback of filamentous phage is its size. The length of an intact phage particle is 930 nm and it contains an ssDNA of 6400 bp. 2800 copies of the major coat protein form a “fish scale” cover over most of the virion DNA, whereas five copies of pIII, which has been the major protein used for library display, and five copies of pVI are located at one end of the filamentous virion. There is no doubt that in order to improve the quality of filamentous phage libraries, the size of phage should be drastically reduced. Comprehensive research on the phage life cycle and its structure will lead us to the construction of miniature phage and to other methods that will enable an in vivo expanding of the library repertoire as well as to binding-induced specific clone-proliferation.     

Rapid evolution of peptide and protein binding properties in vitro.

A significant bottleneck in protein engineering arises from the problem of identifying particular molecules with new functions from a potentially enormous range of peptide or protein variants. Two areas of emerging technology, phage display and multiple peptide synthesis, provide new means of screening huge libraries in vitro for novel binding properties. This review is also published in Current Opinion in Structural Biology 1992, 2:597-604.     

Display of biologically functional insecticidal toxin on the surface of lambda phage

The successful use of Bacillus thuringiensis insecticidal toxins to control agricultural pests could be undermined by the evolution of insect resistance. Under selection pressure in the laboratory, a number of insects have gained resistance to the toxins, and several cases of resistance in the diamondback moth have been reported from the field. The use of protein engineering to develop novel toxins active against resistant insects could offer a solution to this problem. The display of proteins on the surface of phages has been shown to be a powerful technology to search for proteins with new characteristics from combinatorial libraries. However, this potential of phage display to develop Cry toxins with new binding properties and new target specificities has hitherto not been realized because of the failure of displayed Cry toxins to bind their natural receptors. In this work we describe the construction of a display system in which the Cry1Ac toxin is fused to the amino terminus of the capsid protein D of bacteriophage lambda. The resultant phage was viable and infectious, and the displayed toxin interacted successfully with its natural receptor.     

Surface presentation of protein epitopes using bacteriophage expression systems.

Vast libraries of filamentous phage expression vectors that display foreign (poly)peptides on the virion surface can be screened by affinity-purifying those phage whose displayed foreign peptide binds to an antibody or another binding protein. Present libraries display only short random peptides, but work is presently underway to create libraries displaying antibodies with a great diversity of binding specificities.     

A phage display system for studying the sequence determinants of protein folding.

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.     

SiteLight: binding-site prediction using phage display libraries

Phage display enables the presentation of a large number of peptides on the surface of phage particles. Such libraries can be tested for binding to target molecules of interest by means of affinity selection. Here we present SiteLight, a novel computational tool for binding site prediction using phage display libraries. SiteLight is an algorithm that maps the 1D peptide library onto a three-dimensional (3D) protein surface. It is applicable to complexes made up of a protein Template and any type of molecule termed Target. Given the three-dimensional structure of a Template and a collection of sequences derived from biopanning against the Target, the Template interaction site with the Target is predicted. We have created a large diverse data set for assessing the ability of SiteLight to correctly predict binding sites. SiteLight predictive mapping enables discrimination between the binding and nonbinding parts of the surface. This prediction can be used to effectively reduce the surface by 75% without excluding the binding site. In 63% of the cases we have tested, there is at least one binding site prediction that overlaps the interface by at least 50%. These results suggest the applicability of phage display libraries for automated binding site prediction on three-dimensional structures. For most effective binding site prediction we propose using a random phage display library twice, to scan both binding partners of a given complex. The derived peptides are mapped to the other binding partner (now used as a Template). Here, the surface of each partner is reduced by 75%, focusing their relative positions with respect to each other significantly. Such information can be utilized to improve docking algorithms and scoring functions.     

Use of the arabinose p(bad) promoter for tightly regulated display of proteins on bacteriophage

Phage display is a widely used method to optimize the binding characteristics of protein-ligand interactions. In addition, it has been used to clone genes from genomic and cDNA libraries based on their ligand-binding characteristics. One difficulty often encountered when expressing heterologous proteins by phage display is the toxicity of the protein on the Escherichia coli host. Previous studies have shown that heterologous protein expression can be tightly controlled using plasmids with the P(BAD) promoter of the arabinose operon of E. coli, and the araC gene, which is both a positive and negative regulator of the promoter. We constructed a set of phage display vectors that utilize the P(BAD) promoter to control the expression of proteins on the surface of the M13 bacteriophage. These vectors exhibit tightly controlled expression of proteins on the surface of the phage. In addition, the amount of protein displayed on the phage is modulated by the amount of arabinose present in the growth medium during phage propagation. This may be useful for altering the stringency of binding enrichment during phage display.     

Design and synthesis of reagents for phage display screening of dehalogenases

Bifunctional molecules containing both a biotin and a substrate unit have been designed and synthesized for phage display screening of mutant libraries of haloalkane dehalogenase enzymes. The molecules were assembled using a convergent modular synthetic strategy. One molecule was synthesized to evaluate the concept of covalent capture and a second for screening of phage libraries for enantioselectivity.     

New perspective for phage display as an efficient and versatile technology of functional proteomics

Phage display with antibody libraries has been widely used with versatile applications. However, phage display with cDNA libraries is rare and inefficient. Because of uncontrollable reading frames and stop codons in cDNA repertoires, high percentage of phage clones identified from conventional cDNA libraries are non-open reading frames (non-ORFs) encoding unnatural short peptides with minimal implications in protein networks. Consequently, phage display has not been used as a technology of functional proteomics to elucidate protein–protein interactions like yeast two-hybrid system and mass spectrometry-based technologies. Several strategies, including C-terminal display and ORF cDNA libraries, have been explored to circumvent the technical problem. The accumulative endeavors eventually led to the efficient elucidation of a large number of tubby- and phosphatidylserine-binding proteins in recent studies by ORF phage display with minimal reading frame issue. ORF phage display inherits all the versatile applications of antibody phage display, but enables efficient identification of real endogenous proteins with efficiency, sensitivity, and accuracy comparable to other technologies of functional proteomics. Its ELISA-like procedure can be conveniently adapted by individual laboratories or fully automated for high-throughput screening. Thus, ORF phage display is an efficient, sensitive, versatile, and convenient technology of functional proteomics for elucidation of global and pathway-specific protein–protein interactions, disease mechanisms, or therapeutic targets.     

Display of Biologically Functional Insecticidal Toxin on the Surface of λ Phage

The successful use of Bacillus thuringiensis insecticidal toxins to control agricultural pests could be undermined by the evolution of insect resistance. Under selection pressure in the laboratory, a number of insects have gained resistance to the toxins, and several cases of resistance in the diamondback moth have been reported from the field. The use of protein engineering to develop novel toxins active against resistant insects could offer a solution to this problem. The display of proteins on the surface of phages has been shown to be a powerful technology to search for proteins with new characteristics from combinatorial libraries. However, this potential of phage display to develop Cry toxins with new binding properties and new target specificities has hitherto not been realized because of the failure of displayed Cry toxins to bind their natural receptors. In this work we describe the construction of a display system in which the Cry1Ac toxin is fused to the amino terminus of the capsid protein D of bacteriophage lambda. The resultant phage was viable and infectious, and the displayed toxin interacted successfully with its natural receptor.     

Identification of novel prostate-specific antigen-binding peptides modulating its enzyme activity.

Prostate-specific antigen (PSA) is a serine protease with highly prostate-specific expression. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. PSA dissolves the seminal gel forming after ejaculation. It has been suggested to mediate invasion and metastasis of prostate cancer but also to exert antiangiogenic activity. We have identified peptides specific for PSA by screening cyclic phage display peptide libraries. PSA-binding peptides were isolated from four different libraries and produced as a fusion protein with glutathione S-transferase (GST). The phage and fusion proteins were shown to bind to PSA specifically as indicated by lack of binding to other serine proteinases. A peptide with four cysteines showed the highest affinity for PSA. Zn2+, an inhibitor of PSA activity, increased the affinity of the peptides to PSA. The binding specificity was characterized by cross-inhibition using monoclonal anti-PSA antibodies of known epitope specificities. The peptides bound to the same region as mAbs specific for free PSA indicating that they bind close to the active site of the enzyme. The peptides enhanced the enzyme activity of PSA against a chromogenic substrate. These results show that peptides binding to PSA and modulating its enzyme activity can be developed by phage display technique. The peptides have the potential to be used for identification of PSA variants and for imaging and targeting of prostatic tumors.     

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BACKGROUND: The development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised. METHODS: P. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database. RESULTS: Following four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library. CONCLUSION: The construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.     

噬菌体展示技术发展

噬菌体表面展示技术是一种将外源蛋白或抗体可变区与噬菌体表面特定蛋白质融合并展示于其表面,构建蛋白质或抗体库,并从中筛选特异蛋白质或抗体的基因工程技术。随着该项技术的不断完善和发展,噬菌体展示技术已被广泛应用于生命科学研究的不同领域,并显示了良好的应用前景。     

Selection of proteins and peptides from libraries displayed on filamentous bacteriophage

This article attempts to review recent developments in the rapidly developing field of phage display libraries. The current state of peptide, antibody, and cDNA libraries, as well as current and future applications of phage display libraries are discussed. The main focus of the article is on the methods for selecting binding ligands against targets in a variety of different formats. These include solid phase and in-solution selection methods, and the strategies used to select for higher affinity, and binding ligands ampure and cellular target proteins.     

噬菌体展示技术发展

噬菌体表面展示技术是一种将外源蛋白或抗体可变区与噬菌体表面特定蛋白质融合并展示于其表面,构建蛋白质或抗体库,并从中筛选特异蛋白质或抗体的基因工程技术。随着该项技术的不断完善和发展,噬菌体展示技术已被广泛应用于生命科学研究的不同领域,并显示了良好的应用前景。