Engineering a peptide epitope display system on filamentous bacteriophage

Abstract: The genome of bacteriophage fd has been engineered to allow foreign amino acid sequences to be displayed in the exposed N-terminal segment of the major coat protein in the virus particle: small peptides can be encoded directly; larger peptides are encoded in hybrid virions, in which wild-type coat protein subunits are interspersed with coat proteins displaying the foreign peptides. Biophysical techniques, such as X-ray diffraction, indicate that the inclusion of the peptides can be achieved without significant disturbance to the helical parameters that define the protein—protein interactions in the assembled virion and the exposure of the peptides can be verified by analysing the susceptibility to attack by proteolytic enzymes. Peptide sequences from the V3 loop of the surface glycoprotein gp120 of HIV-1 strain MN (HIV-1_MN) displayed in this way are remarkably effective structural mimics of the natural epitope. They are recognised by human HIV antisera and evoke high titres of virus-neutralizing antibodies in mice. Antibody production is stimulated by simultaneous inoculation with T cell epitopes similarly displayed on filamentous bacteriophage. The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins. The specificity of the immune response, the ability to recruit helper T cells, the lack of need for external adjuvants and the structural mimicry of defined peptide epitopes, suggest that it will also be an inexpensive and simple route to the production of effective vaccines.     

The bioanalysis of trastuzumab in human serum using precipitate-enhanced ellipsometry

For the bioanalysis of therapeutic monoclonal antibodies in biological matrices, immunoassays—especially enzyme-linked immunosorbent assays (ELISAs)—are the most widely used techniques. Although ELISAs are very sensitive, the obtained sensitivity is not always sufficient. In this study, we have investigated the possibilities of performing a precipitate-enhanced immunoassay (PEIA) with ellipsometric detection for the bioanalysis of the therapeutic monoclonal antibody trastuzumab. Hydrophobic silicon slides were coated with anti-idiotype trastuzumab antibodies. Trastuzumab in serum samples could bind to this primary catcher, and biotinylated anti-idiotype antibodies were used for detection. After binding of streptavidin-poly-horseradish peroxidase (HRP), the precipitating substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB) was added. Precipitation speed was analyzed using a novel prototype eight-cell ellipsometer, and calibration curves were obtained by plotting this speed versus the trastuzumab concentration. Results demonstrate that the PEIA is at least four times more sensitive than the same ELISA using the chromogenic substrate 3,5,3′,5′-tetramethylbenzidine (TMB) instead of precipitating DAB. The calibration range of the assay is 11 to 700 pg/ml. Serum samples are diluted 10 times prior to incubation corresponding to 110 to 7000 pg/ml in undiluted serum. Validation results demonstrate that these low concentrations can be analyzed accurately and precisely. In addition, samples of a patient treated with trastuzumab were analyzed with both the PEIA and the ELISA. Results demonstrate excellent correlation (r = 0.984) between the methods. Thus, when more sensitivity is required than in a conventional immunoassay, a PEIA with ellipsometric detection may be a useful alternative. The prototype ellipsometer is still in development, and from the data obtained in this study, improvements will be implemented.     

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.     

Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor

Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.     

Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA

Bacteriophage fd gene VIII, which encodes the major capsid protein, was mutated to convert the serine residue at position 47 to a lysine residue (S47K), thereby increasing the number of positively charged residues in the C-terminal region of the protein from four to five. The S47K coat protein underwent correct membrane insertion and processing but could not encapsidate the viral DNA, nor was it incorporated detectably with wild-type coat proteins into hybrid bacteriophage particles. However, hybrid virions could be constructed from the S47K coat protein and a second mutant coat protein, K48Q, the latter containing only three lysine residues in its C-terminal region. K48Q phage particles are approximately 35% longer than wild-type. Introducing the S47K protein shortened these particles, the S47K/K48Q hybrids exhibiting a range of lengths between those of K48Q and wild-type. These results indicate that filamentous bacteriophage length (and the DNA packaging underlying it) are regulated by unusually flexible electrostatic interactions between the C-terminal domain of the coat protein and the DNA. They strongly suggest that wild-type bacteriophage fd makes optimal use of the minimum number of coat protein subunits to package the DNA compactly.     

VLPs Displaying a Single L2 Epitope Induce Broadly Cross-Neutralizing Antibodies against Human Papillomavirus

Background

Virus-like Particles (VLPs) display can be used to increase the immunogenicity of heterologous antigens. Here, we report the use of a bacteriophage MS2-based VLP display platform to develop a monovalent vaccine targeting a broadly neutralizing epitope in the minor capsid protein human papillomavirus (HPV) that provides broad protection from diverse HPV types in a mouse pseudovirus infection model.

Methodology/Principal Findings

Peptides spanning a previously described cross-neutralizing epitope from HPV type 16 were genetically inserted at the N-terminus of MS2 bacteriophage coat protein. Three of the four recombinant L2-coat proteins assembled into VLPs. L2-VLPs elicited high-titer anti-L2 antibodies in mice, similar to recombinant VLPs that we had previously made in which the L2 peptide was displayed on a surface-exposed loop on VLPs of a related bacteriophage, PP7. Somewhat surprisingly, L2-MS2 VLPs elicited antibodies that were much more broadly cross-reactive with L2 peptides from diverse HPV isolates than L2-PP7 VLPs. Similarly, mice immunized with L2-MS2 VLPs were protected from genital and cutaneous infection by highly diverse HPV pseudovirus types.

Conclusion/Significance

We show that peptides can be displayed in a highly immunogenic fashion at the N-terminus of MS2 coat protein VLPs. A VLP-based vaccine targeting HPV L2 elicits broadly cross-reactive and cross-protective antibodies to heterologous HPV types. L2-VLPs could serve as the basis of a broadly protective second generation HPV vaccine.     

Replication of bacteriophage f1: A complex containing gene II protein in gene V mutant-infected bacteria

A dense complex has been isolated from bacteria infected with gene V amber mutant f 1 bacteriophage. The major protein in this complex is the f 1 bacteriophage-specific gene II protein. Other proteins in the complex include the f 1 bacteriophage coat protein and proteins which migrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with the f1 bacteriophage-specific gene III, gene IV and X protein. A protein of approximately 20,000 M_r is also present in the complex. Examination of bacteria infected with gene V mutant f1 bacteriophage revealed the complex as a densely staining amorphous body which appears to be associated with the cytoplasmic membrane. Bacteria infected with f1 bacteriophage that contain amber mutations in genes other than gene V do not contain this complex.     

Quantitative analysis of the bacteriophage Qβ infection cycle

In this study, the infection cycle of bacteriophage Qβ was investigated. Adsorption of bacteriophage Qβ to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4 × 10^− 10 ml/cells/min. In infected cells, approximately 130 molecules of β-subunit and 2 × 10⁵ molecules of coat protein were translated in 15 min. Replication of Qβ RNA proceeded in 2 steps—an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5 × 10⁵ molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.     

Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat

In the initial stage of ascidian fertilization sequential sperm–egg coat interactions assure successful species-specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio-iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non-radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration-dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg-binding activity. Three biotinylated proteins (66kDa, 120kDa and 140kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120kDa protein possesses species-specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species-specific recognition in P. mammillata .     

Probing the kinetics of formation of the bacteriophage MS2 translational operator complex: identification of a protein conformer unable to bind RNA

We have investigated the kinetics of complex formation between bacteriophage MS2 coat protein subunits and synthetic RNA fragments encompassing the natural translational operator site, or the consensus sequences of three distinct RNA aptamer families, which are known to bind to the same site on the protein. Reactions were assayed using stopped-flow fluorescence spectroscopy and either the intrinsic tryptophan fluorescence of the protein or the signals from RNA fragments site-specifically substituted with the fluorescent adenosine analogue 2'-deoxy, 2-aminopurine. The kinetics observed were independent of the fluorophore being monitored or its position within the complex, indicating that the data report global events occurring during complex formation. Competition assays show that the complex being formed consists of a single coat protein dimer and one RNA molecule. The binding reaction is at least biphasic. The faster phase, constituting 80-85 % of the amplitude, is a largely diffusion driven RNA-protein interaction (k1 approximately 2x10(9) M(-1) s(-1)). The salt dependence of the forward reaction and the similarities of the on-rates of lower-affinity RNA fragments are consistent with a diffusion-controlled step dominated by electrostatic steering. The slower phase is independent of reactant concentration, and appears to correspond to isomerisation of the coat protein subunit(s) prior to RNA binding (k(iso) approximately 0.23 s(-1)). Measurements with a coat protein mutant (Pro78Asn) show that this phase is not due to cis-trans isomerisation at this residue. The conformational changes in the protein ligand during formation of an RNA-protein complex might play a role in the triggering of capsid self-assembly and a model for this is discussed.     

Development of an ELISA system for tick-borne encephalitis virus infection in rodents

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. An epizootiological survey of wild rodents is effective to detect TBE virus-endemic areas; however, limited serological diagnostic methods are available to detect anti-TBE virus antibodies in wild rodents. In this study, ELISAs for the detection of rodent antibodies against the TBE virus were developed using two recombinant proteins, domain III of the E protein (EdIII) and subviral particles (SPs), as the antigens. As compared with the neutralization test, the ELISA using EdIII had 77.1% sensitivity and 80.0% specificity, and the ELISA using SPs had 91.4% sensitivity and 100% specificity. Furthermore, when the ELISAs were applied to the epizootiological survey in the TBE virus-endemic area, both of the ELISAs was able to detect wild rodents with TBE virus-specific antibodies. This is the first study to show that ELISAs using recombinant antigens can be safe and useful in the detection of TBE virus-infected wild rodents in epizootiological research.     

Tethering of proteins to RNAs using the bovine immunodeficiency virus-Tat peptide and BIV-TAR RNA

To study the functions of RNA-binding proteins independent of their RNA-binding activity, tethering methods have been developed, based on the use of the RNA-binding domain of a well-characterized RNA-binding protein and its target RNA. Two bacteriophage proteins have mainly been used as tethers: the MS2 coat protein and the lambda N protein. Here we report an alternative system using the Tat (trans-activator) peptide from the bovine immunodeficiency virus (BIV), which binds to BIV-TAR (trans-activation response) RNA. We demonstrate the usefulness of this system by applying it to the analysis of the TNRC6B protein, a component of the microRNA-induced silencing complex.     

Properties of the ribonucleic acid bacteriophage ZIK/1 coat protein and its synthesis in an Escherichia coli cell-free system

The coat protein subunit of the RNA bacteriophage ZIK/1 has a molecular weight of 12100 and does not contain histidine, methionine and cysteine. The amino acid composition of the coat protein is different from that of other RNA bacteriophage coat proteins. Bacteriophage ZIK/1 belongs to a class of RNA bacteriophages distinct from the f2 type, which lack histidine in their coat proteins, and the Qβ type, which lack histidine and methionine. Bacteriophage ZIK/1 RNA is an efficient template in the Escherichia coli cell-free system producing coat protein as the major product and a number of non-coat proteins. This result is similar to that obtained with RNA from f2-type bacteriophages. It is probable that the genomes of RNA bacteriophages are structurally similar and that differences between the types of RNA bacteriophage arise from minor differences in RNA sequence.     

YidC-driven membrane insertion of single fluorescent Pf3 coat proteins

The membrane insertion of single bacteriophage Pf3 coat proteins was observed by confocal fluorescence microscopy. Within seconds after addition of the purified and fluorescently labeled protein to liposomes or proteoliposomes containing the purified and reconstituted membrane insertase YidC of Escherichia coli, the translocation of the labeled residue was detected. The 50-amino-acid-long Pf3 coat protein was labeled with Atto520 and inserted into the proteoliposomes. Translocation of the dye into the proteoliposome was revealed by quenching the fluorescence outside of the vesicles. This allowed us to distinguish single Pf3 coat proteins that only bound to the surface of the liposomes from proteins that had inserted into the bilayer and translocated the dye into the lumen. The Pf3 coat protein required the presence of the YidC membrane insertase, whereas mutants that have a membrane-spanning region with an increased hydrophobicity were autonomously inserted into the liposomes without YidC.     

The protein capsid of filamentous bacteriophage PH75 from Thermus thermophilus

The PH75 strain of filamentous bacteriophage (Inovirus) grows in the thermophilic bacterium Thermus thermophilus at 70 degrees C. We have characterized the viral DNA and determined the amino acid sequence of the major coat protein, p8. The p8 protein is synthesized without a leader sequence, like that of bacteriophage Pf3 but unlike that of bacteriophage Pf1, both of which grow in the mesophile Pseudomonas aeruginosa. X-ray diffraction patterns from ordered fibres of the PH75 virion are similar to those from bacteriophages Pf1 and Pf3, indicating that the protein capsid of the PH75 virion has the same helix symmetry and subunit shape, even though the primary structures of the major coat proteins are quite different and the virions assemble at very different temperatures. We have used this information to build a molecular model of the PH75 protein capsid based on that of Pf1, and refined the model by simulated annealing, using fibre diffraction data extending to 2.4 A resolution in the meridional direction and to 3.1 A resolution in the equatorial direction. The common design may reflect a fundamental motif of alpha-helix packing, although differences exist in the DNA packaging and in the means of insertion of the major coat protein of these filamentous bacteriophages into the membrane of the host bacterial cell. These may reflect differences in the assembly mechanisms of the virions.     

The RNA binding site of bacteriophage MS2 coat protein.

The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.     

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.     

Insights into virus evolution and membrane biogenesis from the structure of the marine lipid-containing bacteriophage PM2

Recent, primarily structural observations indicate that related viruses, harboring no sequence similarity, infect hosts of different domains of life. One such clade of viruses, defined by common capsid architecture and coat protein fold, is the so-called PRD1-adenovirus lineage. Here we report the structure of the marine lipid-containing bacteriophage PM2 determined by crystallographic analyses of the entire approximately 45 MDa virion and of the outer coat proteins P1 and P2, revealing PM2 to be a primeval member of the PRD1-adenovirus lineage with an icosahedral shell and canonical double beta barrel major coat protein. The view of the lipid bilayer, richly decorated with membrane proteins, constitutes a rare visualization of an in vivo membrane. The viral membrane proteins P3 and P6 are organized into a lattice, suggesting a possible assembly pathway to produce the mature virus.     

Adsorption protein of the bacteriophage fd: isolation, molecular properties, and location in the virus.

The adsorption (minor coat) protein of the bacteriophage fd has been implicated to function in several steps of viral morphogenesis. The protein has been purified by sodium dodecyl sulfate gel filtration after dissociation of the virus. The adsorption protein preparation was estimated to have less than 5% contamination by analysis on sodium dodecyl sulfate-polyacrylamide gels and by the results of semiquantitative dansyl-Edman degradation. The amino-terminal sequence of the adsorption protein is H2N-Ala-Glx-Thr-Val-Glx-Ser-Pro-Leu-Pro-. Carboxypeptidase A plus B digestion of the protein under a variety of denaturing conditions did not release any amino acids. There are 3-4 adsorption proteins per virion as estimated by the distribution of E114C]leucine between the major and minor coat protein peaks on sodium dodecyl sulfate-polyacrylamide gels. Adsorption protein-specific antibodies were induced in the rabbit and used as electronmicroscopic markers to determine the position of the adsorption proteins in the viral particle. The adsorption proteins were found at only one end of the filamentous viral particles.