Phage Coat Research Articles

Translational Repression and Specific RNA Binding by the Coat Protein of the Pseudomonas Phage PP7

PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Qbeta. Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Qbeta phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nm. Using a genetic system in which coat protein represses translation of a replicase-beta-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified.

Identification of a collagen-binding protein from Necator americanus by using a cDNA-expression phage display library.

A phage display library was made starting from a cDNA library from the hematophagous human parasite Necator americanus. The cDNA library was transferred by polymerase chain reaction (PCR) cloning into phage display vectors (phagemids), using specially designed primers such that proteins would be expressed as fusions with the C-terminal part of the phage coat protein pVI. The vectors used are multicloning site variants of the original pDONG vectors described by Jespers et al. (1995). Electroporation of the ligation mixtures into electrocompetent Escherichia coli TGI cells yielded 3 x 10(8) pG6A, 1.9 x 10(8) pG6B, and 1 x 10(8) pG6C transfectants for N. americanus. The final libraries consisted of a mix of equal numbers of insert-containing phages from the A, B, and C libraries. Selection of phages for binding to human collagen was performed. Four rounds of panning on human collagens I and III resulted in a significant enrichment of collagen-binding phages from the N. americanus libraries. PCR analysis revealed various insert lengths; however, sequence determination indicated that all phages contained the same protein, albeit with different poly-A tail lengths. The encoded protein itself is a 135-amino acid protein (15 kDa), with no apparent homology to any other known protein. Next the protein was recloned into E. coli using the pET-15b-vector. Upon isopropyl-1-thio-beta-D-galactopyranoside induction, the recombinant protein, rNecH1, could be recovered by urea treatment from inclusion bodies. The rNecH1 protein binds to different collagens: human I > rat I > human III = calf skin I in a specific, dose-dependent, and saturable manner.

Phage display combinatorial libraries of short peptides: ligand selection for protein purification

A library of heptapeptides displayed on the surface of filamentous phage M13 was evaluated as a potential source of affinity ligands for the purification of Rhizomucor miehei lipase. Two independent selection (biopanning) protocols were employed: the enzyme was either physically adsorbed on polystyrene or chemically immobilized on small magnetic beads. From screening with the polystyrene-adsorbed lipase it was found that there was a rapid enrichment of the library with “doublet” clones i.e. the phage species which carried two consecutive sequences of heptapeptides, whilst no such clones were observed from the screening using lipase attached to magnetic beads. The binding of the best clones to the enzyme was unambiguously confirmed by ELISA. However the synthetic heptapeptide of identical sequence to the best “monomeric” clone did not act as a satisfactory affinity ligand after immobilization on Sepharose. This indicated that the interaction with lipase was due to both the heptapeptide and the presence of a part of the phage coat protein. This conclusion was further verified by immobilizing the whole phage on the surface of magnetic beads and using the resulting conjugate as an affinity adsorbent. The scope of application of this methodology and the possibility of preparing phage-based affinity materials are briefly discussed.

Exchange of the basic domain of human immunodeficiency virus type 1 Rev for a polyarginine stretch expands the RNA binding specificity, and a minimal arginine cluster is required for optimal RRE RNA binding affinity, nuclear accumulation, and trans-activation.

The Rev regulatory protein of human immunodeficiency virus (HIV) facilitates the nuclear export of unspliced and partially spliced HIV RNAs. Using a Rev:MS2 phage coat protein fusion that could be targeted to bind and activate the Rev-responsive element (RRE) RNA or heterologous MS2 phage operator RNA, we analyzed the role(s) of the arginine-rich RNA binding domain in RNA binding and transactivation. The arginine-rich domain could be functionally replaced by a stretch of nine arginines. However, polyarginine substitutions expanded the RNA binding specificity of the resultant mutant Rev protein. Polyarginine insertions in place of residues 24 to 60 that excised the RNA binding and oligomerization domains of Rev preserved the activation for MS2 RNA, but not for the RRE. A nine-arginine insertion outside of the natural context of the Rev nuclear localization signal domain was incompatible with activation of either RNA target. Insertions of fewer than eight arginines impaired RRE activation. Interrupted lysine clusters and disruption of the arginine stretch with lysine or neutral residues resulted in a similar phenotype. Some of these mutants with a null phenotype for RRE activated the heterologous MS2 RNA target. Under steady-state conditions, mutants that preserved the Rev response for RRE RNA localized to the nuclei; those with poor or no Rev response accumulated mostly in the cytoplasm. Many of the cytoplasmically resident derivatives became nuclear when leptomycin B (LMB) treatment inhibited nuclear export of nuclear export signal-containing proteins. Mutants that had a null activation potential for either RNA target were particularly resistant to LMB treatment. Abbreviated nuclear residence times and differences in RRE binding affinity may have compromised their activation potential for RRE. High-affinity binding to MS2 RNA through the intact coat protein was sufficient to overcome the short nuclear residence times and to facilitate MS2 activation by some derivatives.

Biotechnological applications of phage and cell display

In recent years, the use of surface-display vectors for displaying polypeptides on the surface of bacteriophage and bacteria, combined with in vitro selection technologies, has transformed the way in which we generate and manipulate ligands, such as enzymes, antibodies and peptides. Phage display is based on expressing recombinant proteins or peptides fused to a phage coat protein. Bacterial display is based on expressing recombinant proteins fused to sorting signals that direct their incorporation on the cell surface. In both systems, the genetic information encoding for the displayed molecule is physically linked to its product via the displaying particle. Using these two complementary technologies, we are now able to design repertoires of ligands from scratch and use the power of affinity selection to select those ligands having the desired (biological) properties from a large excess of irrelevant ones. With phage display, tailor-made proteins (fused peptides, antibodies, enzymes, DNA-binding proteins) may be synthesized and selected to acquire the desired catalytic properties or affinity of binding and specificity for in vitro and in vivo diagnosis, for immunotherapy of human disease or for biocatalysis. Bacterial surface display has found a range of applications in the expression of various antigenic determinants, heterologous enzymes, single-chain antibodies, and combinatorial peptide libraries. This review explains the basis of phage and bacterial surface display and discusses the contributions made by these two leading technologies to biotechnological applications. This review focuses mainly on three areas where phage and cell display have had the greatest impact, namely, antibody engineering, enzyme technology and vaccine development.

A Novel Approach and a Novel Mechanism for Stealthing Gene Delivery Vehicles

A Novel Approach and a Novel Mechanism for Stealthing Gene Delivery Vehicles

The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.

The icosahedral T7 phage (diameter approximately 65 nm) displaying random peptides at the carboxy-terminus of the phage coat proteins was used as a model for drug and gene delivery vehicles containing peptide ligands. We found that displayed peptides were recognized by natural antibodies and induced complement activation. Strikingly, the phage inactivation by complement was peptide-specific that implied the existence of numerous natural antibodies with different peptide specificity. Selection of phage that avoided inactivation by complement allowed the identification of peptides that protected the phage by binding to serum proteins. In rat blood, peptides with carboxy-terminal lysine or arginine residues protected the phage against complement-mediated inactivation by binding C-reactive protein. In human serum, a number of protective peptides with tyrosine residues were selected. The recognition of displayed peptides by natural antibodies appears to represent a universal mechanism for activation of complement at sites that contain identical or homologous proteins with exposed carboxy-termini.

Homodimeric peptides displayed by the major coat protein of filamentous phage

Peptide libraries displayed by filamentous bacteriophage have proven a powerful tool for the discovery of novel peptide agonists, antagonists and epitope mimics. Most phage-displayed peptides are fused to the N terminus of either the minor coat protein, pIII, or the major coat protein, pVIII. We report here that peptides containing cysteine residues, displayed as N-terminal fusions to pVIII, can form disulfide-bridged homodimers on the phage coat. Phage clones were randomly selected from libraries containing one or two fixed Cys residues, and surveyed for the presence of peptide-pVIII homodimers by SDS-PAGE analysis that involved pretreatment of the phage with reducing or thiol-modifying agents. For all phage whose recombinant peptide contained a single Cys residue, a significant fraction of the peptide-pVIII molecules were displayed as dimers on the phage coat. The dimeric form was in greater abundance than the monomer in almost all cases in which both forms could be reliably observed. Occasionally, peptides containing two Cys residues also formed dimers. These results indicate that, for a given pVIII-displayed peptide bearing a single Cys residue, a significant fraction of the peptide (>40 %) will dimerize regardless of its sequence; however, sequence constraints probably determine whether all of the peptide will dimerize. Similarly, only occasionally do peptides bearing two Cys residues form intermolecular disulfide bridges instead of intramolecular ones; this indicates that sequence constraints may also determine dimerization versus cyclization. Sucrose-gradient analysis of membranes from cells expressing pVIII fused to a peptide containing a single Cys residue showed that dimeric pVIII is present in the cell prior to its assembly onto phage. A model of the peptide-pVIII homodimer is discussed in light of existing models of the structure and assembly of the phage coat. The unique secondary structures created by the covalent association of peptides on the phage surface suggest a role for homo- and heterodimeric peptide libraries as novel sources of bioactive peptides.

Development and application of cytotoxic T lymphocyte-associated antigen 4 as a protein scaffold for the generation of novel binding ligands

We have explored the possibilities of using human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) as a single immunoglobulin fold-based scaffold for the generation of novel binding ligands. To obtain a suitable protein library selection system, the extracellular domain of CTLA-4 was first displayed on the surface of a filamentous phage as a fusion product of the phage coat protein p3. CTLA-4 was shown to be functionally intact by binding to its natural ligands B7-1 (CD80) and B7-2 (CD86) both in vitro and in situ. Secondly, the complementarity determining region 3 (CDR3) loop of the CTLA-4 extracellular domain was evaluated as a permissive site. We replaced the nine amino acid CDR3-like loop of CTLA-4 with the sequence XXX-RGD-XXX (where X represents any amino acid). Using phage display we selected several CTLA-4-based variants capable of binding to human αvβ3 integrin, one of which showed binding to integrins in situ. To explore the construction of bispecific molecules we also evaluated one other potential permissive site diametrically opposite the natural CDR-like loops, which was found to be tolerant of peptide insertion. Our data suggest that CTLA-4 is a suitable human scaffold for engineering single-domain molecules with one or possibly more binding specificities.

Design and evolution of artificial M13 coat proteins

Using simple design and selective pressure, we have evolved an artificial M13 bacteriophage coat protein. M13 coat proteins first reside in the bacterial inner membrane and subsequently surround the DNA core of the assembled virus. The artificial coat protein (ACP) was designed and evolved to mimic both functions of the natural M13 coat proteins, but with an inverted orientation. ACP is a non-functional coat protein because it is not required for the production of phage particles. Instead, it incorporates into a phage coat which still requires all the natural coat proteins for structural integrity. In contrast with other M13 coat proteins, which can display polypeptides as amino-terminal fusions, ACP permits the carboxy-terminal display of large polypeptides. The results suggest that viruses can co-opt host membrane proteins to acquire new coat proteins and thus new functions. In particular, M13 bacteriophage can be engineered for new functions, such as carboxy-terminal phage display.

Recombinant expression and neutralizing activity of an MHC class II binding epitope of toxic shock syndrome toxin-1

Toxic shock syndrome (TSS) is caused by the staphylococcal superantigen, TSST-1. The MHC class II binding domain of TSST-1 containing a conserved sequence with other related staphylococcal enterotoxins, comprising TSST-1 residues 47–64 [(T(47–64)], was expressed as a fusion protein with either glutathione- S-transferase (GST 47–64), filamentous phage coat protein (pIII 47–64), or E. coli outer membrane porin protein (OprF 47–64), or synthesized as a peptide conjugated to bovine serum albumin, BSA 47–64. GST 47–64, OprF 47–64 and BSA 47–64, but not pIII 47–64, all induced high-titer T(47–64)-specific antibodies in Balb/c mice. However, only anti-GST 47–64 antibodies inhibited 125I-TSST-1 binding to MHC class II and abrogated TSST-1-induced T cell mitogenesis and TNF α secretion in human peripheral blood mononuclear cells. Purified GST 47–64 also inhibited 125I-TSST-1 binding in a dose-dependent manner. These findings suggest that GST 47–64 may have potential as a recombinant peptide vaccine or TSST-1 receptor inhibitor against TSS.

High copy display of large proteins on phage for functional selections

We have isolated mutations in the major coat protein P8 of M13 phage that greatly increase the surface display of monomeric or oligomeric proteins. The monomeric protein, human growth hormone (hGH), was fused to the N terminus of P8; libraries of P8 variants were constructed and variants that increased hGH display were selected by binding to the extracellular domain of the hGH receptor. The hGH-P8 fusion protein was found to be extremely tolerant of mutations, and a number of P8 variants were found that increased display to levels that improved detection of the hGH-P8 fusion by almost 100-fold. The increased display likely results from better accommodation of the hGH-P8 fusion protein in the phage coat. Using this high copy display format, it was possible for the first time to detect variants of hGH with very weak affinities for the hGHbp (Kd>1 μM). The display of a tetrameric protein, streptavidin (≈50 kDa), was also increased, suggesting the approach may be general to many proteins. The initial product of a natural or invented selection from a naive library is often a weakly functioning protein. These improvements in high copy display should facilitate the broader goal for selection of proteins with novel functions.

Mutational analysis of the major coat protein of M13 identifies residues that control protein display.

We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487-495). Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus. Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities.

Interdomain interactions within the gene 3 protein of filamentous phage

Infection of Escherichia coli by filamentous phage fd is mediated by the phage gene 3 protein (g3p). The g3p consists of three domains (g3p-D1, D2 and D3) linked by flexible glycine-rich linkers. All three domains are indispensable for phage infectivity; the g3p-D1 domain binds to the TolA receptor presumably at the inner face of the outer membrane, the g3p-D2 domain to the F-pilus and the g3p-D3 domain anchors g3p to the phage coat. The N-terminal domains g3p-D1 and D2 interact with each other; this interaction is abrogated by binding of g3p-D2 to the F-pilus leading to the release of g3p-D1 to bind to TolA. Here, using phages with deletions in g3p, we have discovered a specific interaction between the two N-terminal domains and g3p-D3, the C-terminal domain of g3p. We propose that these interdomain interactions within g3p lead to a compact and stable organisation when displayed on the phage tip, but that during infection, this compact state must be unraveled.

Display of Ras on filamentous phage through cysteine replacement

Phage display technology has been used in a variety of contexts to understand and manipulate biomolecular interactions between proteins and other biomolecules. In this paper we describe the establishment of a phage display system for elucidation of the interactions between the GTPase Ras and its panel of effectors. It is shown how technical problems associated with phage display of a protein with unpaired cysteines, likely to be caused by the oxidizing environment of the bacterial periplasm into which the protein is directed, can be overcome by cysteine replacement based on functional and structural studies. First, the catalytic domain (residues 1–166) of mammalian H-Ras (Ras) was observed to be displayed on phage in an incorrect conformation not detectable by antibodies recognizing conformational epitopes on Ras. Although truncation of the phage coat protein used as fusion partner (g3p) resulted in minor improvements in the display, Ras was tailored for phage display by cysteine replacement. By replacing the three cysteines at positions 51, 80 and 118 of Ras with the corresponding residues in Saccharomyces cerevisiae RAS1, the resulting fusion-phage is recognized by the conformation-dependent anti-Ras antibodies. Furthermore, display of cysteine-free Ras is demonstrated by GTP-analogue dependent binding to the Ras-binding domain of the Ras-effector Raf1. These data pave the way for analysis of Ras-effector interactions using phage display technology yet demonstrate that phage display of proteins with normally reduced cysteines should be approached with caution.

Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd

The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (alpha subunit-internal peptide-beta subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the beta-subunit is covalently linked and the alpha-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.

Isolation of peptides that mimic epitopes on a malarial antigen from random peptide libraries displayed on phage.

The ring-infected erythrocyte surface antigen (RESA) is a dense-granule protein of Plasmodium falciparum which binds to the cytoskeletal structure of the erythrocyte after parasite invasion. It is currently under trial as a vaccine candidate. In an effort to characterize further the antibody responses to this antigen, we have panned two independent libraries of random peptides expressed on the surface of filamentous phage with a monoclonal antibody (MAb 18/2) against RESA. One library consisted of a potentially constrained 17-mer peptide fused with the gpVIII phage coat protein, and the other displayed an unconstrained 15-mer as a fusion with the minor phage coat protein gpIII. Several rounds of biopanning resulted in enrichment from both libraries clones that interacted specifically with MAb 18/2 in protein-blotting and enzyme-linked immunosorbent assay experiments. Nucleotide sequencing of the random oligonucleotide insert revealed a common predominant motif: (S/T)AVDD. Several other clones had related but degenerate motifs. Thus, a monoclonal antibody against a malarial antigen can select common mimotopes from different random peptide libraries. We envisage many uses for this technology in malaria research.

Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd

The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (α subunit-internal peptide-β subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the β-subunit is covalently linked and the α-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.

Selection of specific phage from display libraries: monoclonal antibody against VCS M13 helper phage coat protein III (gIIIp).

Screening of specific phage is often hampered by nonspecific binding either of the VCS M13 helper phage to the solid phase absorbent or to the polyclonal antibodies used for selection. The former is improved by increasing the stringency for selection. However, the available polyclonal anti-VCS M13 antibodies often react with immobilized antigen nonspecifically, making it difficult to distinguish between positive and negative clones. To improve this selection process, a monoclonal antibody (MAb) was produced which recognizes ligand-coat protein three (gIIIp) on the helper phage VCS M13. This MAb is highly sensitive and specific, and it is useful for selecting relevant clones. This reagent should find widespread application in identifying interactive clones from a variety of phage display libraries.

Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 Å: evidence for conformational lability

Infection of Escherichia coli by filamentous bacteriophages is mediated by the minor phage coat protein g3p and involves two distinct cellular receptors, the F′ pilus and the periplasmic protein TolA. Recently we have shown that the two receptors are contacted in a sequential manner, such that binding of TolA by the N-terminal domain g3p-D1 is conditional on a primary interaction of the second g3p domain D2 with the F′ pilus. In order to better understand this process, we have solved the crystal structure of the g3p-D1D2 fragment (residues 2–217) from filamentous phage fd to 1.9 Å resolution and compared it to the recently published structure of the same fragment from the related Ff phage M13. While the structure of individual domains D1 and D2 of the two phages are very similar (rms < 0.7 Å), there is comparatively poor agreement for the overall D1D2 structure (rms > 1.2 Å). This is due to an apparent movement of domain D2 with respect to D1, which results in a widening of the inter-domain groove compared to the structure of the homologous M13 protein. The movement of D2 can be described as a rigid-body rotation around a hinge located at the end of a short anti-parallel beta-sheet connecting domains D1 and D2. Structural flexibility of at least parts of the D1D2 structure was also suggested by studying the thermal unfolding of g3p: the TolA binding site on D1, while fully blocked by D2 at 37 °C, becomes accessible after incubation at temperatures as low as 45 °C. Our results support a model for the early steps of phage infection whereby exposure of the coreceptor binding site on D1 is facilitated by a conformational change in the D1D2 structure, which in vivo is induced by binding to the F′ pilus on the host cell and which can be mimicked in vitro by thermal unfolding.