A novel method for viral display of ER membrane proteins on budded baculovirus

The baculovirus expression system has been used to express large quantities of various proteins, including membrane receptors. Here, we reveal a novel property of this expression system to be that certain membrane proteins can be displayed on the budded virus itself. We introduced the genes encoding sterol regulatory element-binding protein-2 (SREBP-2) or SREBP cleavage-activating protein (SCAP), important integral membrane proteins of the endoplasmic reticulum (ER) and/or the Golgi apparatus related to cellular cholesterol regulation, into a baculovirus vector. When insect cells were infected with SREBP-2 or SCAP recombinant viruses, it was found that these ER membrane proteins appeared on the budded baculovirus in addition to the host cell membrane fraction. Compared to proteins expressed on the cell membrane, membrane proteins displayed on virus exhibited both less aggregation and less degradation upon immunoblotting. Using this viral displayed SCAP as the screening antigen, we then generated a new monoclonal antibody specific against SCAP, which was useful for immunological localization studies. This system, which takes advantage of the viral display of membrane proteins, should prove to be a powerful additional tool for postgenomic protein analysis.     

Yeast surface display of full-length human microtubule-associated protein tau

Microtubule-associated protein tau is an intrinsically disordered, highly soluble protein found primarily in neurons. Under normal conditions, tau regulates the stability of axonal microtubules and intracellular vesicle transport. However, in patients of neurodegeneration such as Alzheimer's disease (AD), tau forms neurofibrillary deposits, which correlates well with the disease progression. Identifying molecular signatures in tau, such as posttranslational modification, truncation, and conformational change has great potential to detect earliest signs of neurodegeneration and develop therapeutic strategies. Here, we show that full-length human tau, including the longest isoform found in the adult brain, can be robustly displayed on the surface of yeast Saccharomyces cerevisiae. Yeast-displayed tau binds to anti-tau antibodies that cover epitopes ranging from the N-terminus to the 4R repeat region. Unlike tau expressed in the yeast cytosol, surface-displayed tau was not phosphorylated at sites found in AD patients (probed by antibodies AT8, AT270, AT180, and PHF-1). However, yeast-displayed tau showed clear binding to paired helical filament (PHF) tau conformation-specific antibodies Alz-50, MC-1, and Tau-2. Although the tau possessed a conformation found in PHFs, oligomerization or aggregation into larger filaments was undetected. Taken together, yeast-displayed tau enables robust measurement of protein interactions and is of particular interest for characterizing conformational change.     

Proteomics on full-length membrane proteins using mass spectrometry

A general technique has been developed that allows rapid mass spectrometric analysis of full-length membrane proteins [Whitelegge, J. P., le Coutre, J., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10695-10698]. Using in-line HPLC electrospray ionization mass spectrometry (LC-MS), different native and recombinant bacterial membrane proteins of up to 61 kDa are characterized. Mass spectrometric data of four entirely different membrane proteins from three bacterial organisms, two transporters, a channel, and a porin protein are presented. In addition to determination of the molecular mass with an accuracy of +/-0.01%, the technique monitors alkylation or oxidation of single Cys residues and errors in deduced amino acid sequences. Finally, using in-line LC-MS, unknown proteins can be identified from solubilized Escherichia coli membranes without prior purification.     

Phage matrix for isolation of glioma cell membrane proteins

Cell-binding ligands for RG2 rat glioma were identified in our recent study from a library of peptides that are displayed as fusion molecules on phage particles. Here, one of the phage clones was used to affinity purify those cell membrane components to which the displayed peptides bind. This phage clone, displaying the ELRGDSLP peptide, was shown to recognize glioma cells specifically in comparison to control phage-expressing peptides of either similar or irrelevant sequences. Blocking experiments with synthetic RGDS peptide demonstrated that the phage-glioma cell recognition occurs via the RGD motif known to be present in many integrin-binding proteins. To form an affinity matrix that would bind to glioma cell membrane molecules, ELRGDSLP phage particles were cross-linked using dextran polymer. Whole cell lysate from RG2 rat glioma cells was passed through the matrix, resulting in the isolation of cell membrane components having strong affinity to the peptides on phage and molecules associated with those components. One of the isolated proteins was found to be CD44s, a cell surface adhesion molecule involved in glioma cell invasion and migration, which likely formed a complex with an RGD-binding integrin. Cell membrane proteins isolated with this innovative approach could be used for the design of cell-specific anticancer treatments.     

Phage display as a novel screening method to identify extracellular proteins

Extracellular proteins are involved in many diverse and essential cell functions and in pathogenic bacteria, and they may also serve as virulence factors. Therefore, there is a need for methods that identify the genes encoding this group of proteins in a bacterial genome. Here, we present such a method based on the phage display technology. A novel gene III-based phagemid vector, pG3DSS, was constructed that lacks the signal sequence which normally orientates the encoded fusion protein to the Escherichia coli cell membrane, where it is assembled into the phage particle. When randomly fragmented DNA is inserted into this vector, only phagemids containing an insert encoding a signal sequence will give rise to phage particles displaying a fusion protein. These phages also display an E-tag epitope in fusion with protein III, which enables isolation of phages displaying a fusion protein, using antibodies against the epitope. From a library constructed from Staphylococcus aureus chromosomal DNA, genes encoding secreted as well as transmembrane proteins were isolated, including adhesins, enzymes and transport proteins.     

Phage display for the production of human monoclonal antibodies against human pathogens

In the last decade an increasing number of antibodies have made their way from the research benchtops into the clinics and many more are currently under clinical trial. Among monoclonal antibody-producing techniques, phage-display is undoubtedly the most effective and versatile. Cloning of the entire humoral repertoire derived from an infected patients into a phage display vector allows not only the simple generation of monoclonal antibodies of desired specificity, but also the molecular dissection of the antibody response itself. Generation of large panels of human monoclonal antibodies against human pathogens could open new perspectives in understanding the interplay between the infectious agent and the infected host providing tools for the prevention and the therapy of human communicable diseases. In this paper the basic principles of the phage-display approach as well as its most recent applications are reviewed.     

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.     

Phage display of functional human TNF-alpha converting enzyme catalytic domain: a rapid method for the production of stabilized proteolytic proteins for assay development and high-throughput screening

The catalytic domain of human tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS). This would address many issues around screening of proteases in this class. The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF-alpha to generate the mature TNF-alpha in vitro. To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF-alpha-specific peptide in vitro. More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS. The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative efficacy of PDT. Both PDT and BET showed a similar specific cleavage profile against the defined substrates. Activity of the BET, however, was stable at 4 degrees C for less than 24 h. In contrast, the PDT exhibited remarkable stability, losing very little activity even after 2 years at 4 degrees C. On the basis of these results, the authors concluded that the phage display system might be a useful tool for expressing proteins that have stability issues related to auto-proteolytic activity. Furthermore, the ease and low cost of large-scale production of phage should make it suitable for assay development and HTS.     

Phage display systems and their applications

Screening phage display libraries of proteins and peptides has, for almost two decades, proven to be a powerful technology for selecting polypeptides with desired biological and physicochemical properties from huge molecular libraries. The scope of phage display applications continues to expand. Recent applications and technical improvements driving further developments in the field of phage display are discussed.     

Phage display reveals multiple contact sites between FhuA, an outer membrane receptor of Escherichia coli, and TonB

The ferric hydroxamate uptake receptor FhuA from Escherichia coli transports siderophores across the outer membrane (OM). TonB-ExbB-ExbD transduces energy from the cytoplasmic membrane to the OM by contacts between TonB and OM receptors that contain the Ton box, a consensus sequence near the N terminus. Although the Ton box is a region of known contact between OM receptors and TonB, our biophysical studies established that TonB binds to FhuA through multiple regions of interaction. Panning of phage-displayed random peptide libraries (Ph.D.-12, Ph.D.-C7C) against TonB identified peptide sequences that specifically interact with TonB. Analyses of these sequences using the Receptor Ligand Contacts (RELIC) suite of programs revealed clusters of multiply aligned peptides that mapped to FhuA. These clusters localized to a continuous periplasm-accessible surface: Ton box/switch helix; cork domain/beta1 strand; and periplasmic turn 8. Guided by such matches, synthetic oligonucleotides corresponding to DNA sequences identical to fhuA were fused to malE; peptides corresponding to the above regions were displayed at the N terminus of E.coli maltose-binding protein (MBP). Purified FhuA peptides fused to MBP bound specifically to TonB by ELISA. Furthermore, they competed with ligand-loaded FhuA for binding to TonB. RELIC also identified clusters of multiply aligned peptides corresponding to the Ton box regions in BtuB, FepA, and FecA; to periplasmic turn 8 in BtuB and FecA; and to periplasmic turns 1 and 2 in FepA. These experimental outcomes identify specific molecular contacts made between TonB and OM receptors that extend beyond the well-characterized Ton box.     

Phage display: practicalities and prospects

Phage display is a molecular technique by which foreign proteins are expressed at the surface of phage particles. Such phage thereby become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed proteins, but also have the capacity to replicate. Using phage display vast numbers of variant nucleotide sequences may be converted into populations of variant peptides and proteins which may be screened for desired properties. It is now some seventeen years since the first demonstration of the feasibility of this technology and the intervening years have seen an explosion in its applications. This review discusses the major uses of phage display including its use for elucidating protein interactions, molecular evolution and for the production of recombinant antibodies.     

The scope of phage display for membrane proteins

Numerous examples of phage display applied to soluble proteins demonstrate the power of the technique for protein engineering, affinity reagent discovery and structure-function studies. Recent reports have expanded phage display to include membrane proteins (MPs). The scope and limitations of MP display remain undefined. Therefore, we report data from the phage display of representative types of membrane-associated proteins including plasma, nuclear, peripheral, single and multipass. The peripheral MP neuromodulin displays robustly with packaging by conventional M13-KO7 helper phage. The monotopic MP Nogo-66 can also display on the phage surface, if packaged by the modified M13-KO7⁺ helper phage. The modified phage coat of KO7⁺ can better mimic the zwitterionic character of the plasma membrane. Four examples of putatively α-helical, integral MPs failed to express as fusions to an anchoring phage coat protein and therefore did not display on the phage surface. However, the β-barrel MPs ShuA (Shigella heme uptake A) and MOMP (major outer membrane protein), which pass through the membrane 22 and 16 times, respectively, can display surprisingly well on the surfaces of both conventional and KO7⁺ phages. The results provide a guide for protein engineering and large-scale mutagenesis enabled by the phage display of MPs.     

High-density functional display of proteins on bacteriophage lambda

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display.This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.     

噬菌体展示肽文库及其应用

肽库是研究与特定靶分子高亲和结合配体的有力工具,肽文库可被分为合成文库和噬菌体肽文库两类,作者综述了噬菌体肽文库及其蛋白质结构域分析定位,疫苗研制及抗原表位分析和药物设计等方面的应用。     

Phage display of random peptide libraries: applications, limits, and potential

The identification of ligands from large biological libraries by phage display has now been used for almost 15 years. Most of the successful reports on high-affinity ligand identification originated from work with different antibody libraries. In contrast, the progress of applying phage display to random peptide libraries was relatively slow. However, in the last few years several improvements have led to an increasing number of published peptide ligands identified by phage display from such libraries and which exhibited good biological activity and high affinity. This review summarizes the current state and the technical progress of the application of random peptide libraries using filamentous phage for ligand identification.     

Biophysical and functional assays for viral membrane fusion peptides

Membrane fusion is a protein catalyzed biophysical reaction that involves the simultaneous intermixing of two phospholipid bilayers and of the aqueous compartments bound by their respective bilayers. In the case of enveloped virus fusogens, short hydrophobic or amphipathic fusion peptides that are components of the larger fusion complex are essential for the membrane merger event. The process of cell–cell membrane fusion and syncytium formation induced by the nonenveloped fusogenic orthoreoviruses is driven by the Fusion-Associated Small Transmembrane (FAST) proteins, which are similarly dependent on the action of fusion peptides. In this article, we describe some simple methods for the biophysical characterization of viral membrane fusion peptides. Liposomes serve as an ideal model system for characterizing peptide–membrane interactions because their size, shape and composition can be readily manipulated. We present details of fluorescence assays used to elucidate the kinetics of membrane fusion as well as complimentary assays used to characterize peptide-induced liposome binding and aggregation.     

Phage display of catalytically active staphylococcal nuclease

Staphylococcal nuclease (SNase), a 14 kD enzyme that catalyzes the hydrolysis of single- and double-stranded nucleic acid, was fused to the N-terminus of the gene III (pIII) protein of filamentous phage fdtet. The SNase-pIII protein is infective and the catalyzes DNA hydrolysis, demonstrating that functional SNase can be displayed on the phage surface.