Staphylococcus aureus Pathogenicity Island DNA Is Packaged in Particles Composed of Phage Proteins
María Ángeles Tormo‐MásMaría D. FerrerElisa MaiquesCarles ÚbedaLaura SelvaÍñigo LasaJuan J. CalveteRichard P. NovickJosé R. Penades
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ABSTRACT Staphylococcus aureus pathogenicity islands (SaPIs) have an intimate relationship with temperate staphylococcal phages. During phage growth, SaPIs are induced to replicate and are efficiently encapsidated into special small phage heads commensurate with their size. We have analyzed by amino acid sequencing and mass spectrometry the protein composition of the specific SaPI particles. This has enabled identification of major capsid and tail proteins and a putative portal protein. As expected, all these proteins were phage encoded. Additionally, these analyses suggested the existence of a protein required for the formation of functional phage but not SaPI particles. Mutational analysis demonstrated that the phage proteins identified were involved only in the formation and possibly the function of SaPI or phage particles, having no role in other SaPI or phage functions.Keywords:
Temperateness
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Abstract Monovalent phage display enables a single protein (or peptide) molecule to be presented on the surface of a phage particle. This protein is translated from a phagemid vector as a fusion linked to the natural pIII coat protein in an Escherichia coli cell coinfected with helper phage. The display achieves monova- lency since the fusion protein rarely substitutes for the native pIII protein from the helper phage genome. While display can be highly variable, typically just 0.05-5% of these phages actually display the protein of interest (1).
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Abstract Phage display is a process by which a peptide or a protein is expressed as an exterior fusion to a surface protein of a phage particle. The peptide or protein sequence can be deduced from its encoding DNA sequence that resides in the phage particle or in a transductant. Amplification of the DNA of interest can take place by phage/transductant propagation or by polymerase chain reaction (PCR). By producing large populations of phage particles, each expressing a unique peptide or protein, peptide/protein libraries can be obtained. Peptides or proteins, interacting with defined molecular targets most often proteins can be isolated from such libraries by enrichment through repeated cycles of panning. Hence, phage display can be thought of as a ‘‘search engine’’ of protein^target interactions. The pioneering work of Smith (113) first demonstrated surface display of peptides in filamentous phage fd. This innovation was extended to peptide libraries of fd and M13 (19, 22, 108) and phagemid display was introduced (6). The display of proteins such as antibody domains and combinatorial antibody libraries soon followed (5, 59, 75). From the beginning of the 1990s phage-display-related publications have grown exponentially (128). Several reviews (some general, e.g., 58, 125; and many specialized) are available. There are numerous reports and several laboratory manuals (4) describing development and use of filamentous phage display in identification of peptide or protein interactions with simple organic compounds, antibodies, receptors, etc. Phage display is also a useful tool in protein engineering and directed evolution (44). Then there is the large sector of phage antibody display (64) and the more recent field of immune profiling and its implication for vaccine development (15, 112). Furthermore, complex targets such as cells (92) and whole tissues/organs (91) have been subjected to phage display analysis. These studies explore novel approaches for in vivo homing in gene/drug delivery (78), cancer surveillance/treatment (2, 86, 103), and imaging (127).
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To construct a human phage display single-chain Fv (ScFv) antibody library against Mycobacterium tuberculosis (MTB), for specific ScFv antibody cloning.Total RNA was isolated from the lymphocytes of patients with positive serum antibody against MTB and reverse transcribed into cDNA. The heavy chain and light chain variable region gene of human immunoglobulin were amplified individually by PCR and then assembled into ScFv genes. The ScFv genes were ligated into phagemid pCANTAB5S. The human phage display ScFv library against MTB was constructed by transforming the recombinant phagemid into E. coli TG1 with the presence of helper phage M13K07.The human phage display ScFv library containing 10(7) different clones was constructed successfully.A phage display ScFv library against MTB has been constructed based on the variable region gene of immunoglobulin of the lymphocytes of TB patients and phagemid pCANTAB5S. The specific ScFv antibodies can be screened from this library.
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Phage display is a widely used gene engineering high technology.Through the display of exogenous peptides or proteins fused specific coat protein on the surface of phages,it is possible to construct proteins or peptides libraries and screen interesting proteins,peptides and antibodies successfully.Most commonly used phage display technology is phagemid/helper phage system,in which helper phages are essential for the replication and assembly of phagemid particles.In this review,in combination with the newest research dynamic status,we summarize phagemid/helper phage double-genome system.We mainly emphasized the features and mutation mechanisms of different helper phages.We also made some prospects for the future directions,in the meanwhile,we also expect that our experience can provide some help for further maturity of the technology.
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We present herein a novel method of pIII-based antibody phage display using Hpd3cells—bacterial cells bearing the genome of a gene-III-lacking helper phage (VCSM13d3). A high level of single-chain variable fragments (scFvs) was displayed in the consequent phagemid particles using Hpd3cells to rescue the phagemid encoding scFv-pIII. Hpd3cells considerably improved the specific enrichment factor when used for constructing an immunized antibody library. In addition, using Hpd3cells could overcome pIII resistance and can contribute to the efficient enrichment of specific binding antibodies from a phage display library, thereby increasing the chance of obtaining more diverse antibodies specific for target antigens.
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Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin. However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules. We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage. Ex-phage has a mutant pIII gene that produces a functional wild-type pIII in suppressing Escherichia coli strains but does not make any pIII in non-suppressing E.coli strains. Packaging phagemids encoding antibody-pIII fusion in F+ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13KO7 helper phage. Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.
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A phagemid is a plasmid that contains the origin of replication and packaging signal of a filamentous phage. Following bacterial transformation, a phagemid can be replicated and amplified as a plasmid, using a double-stranded DNA origin of replication, or it can be replicated as single-stranded DNA for packaging into filamentous phage particles. The use of phagemids enables phage display of large proteins, such as antibody fragments. Phagemid pComb3 was among the first phage display vectors used for the generation and selection of antibody libraries in the 50-kDa Fab format, a monovalent proxy of natural antibodies. Affording a robust and versatile tool for more than three decades, phage display vectors of the pComb3 phagemid family have been widely used for the discovery, affinity maturation, and humanization of antibodies in Fab, scFv, and single-domain formats from naive, immune, and synthetic antibody repertoires. In addition, they have been used for broadening phage display to the mining of nonimmunoglobulin repertoires. This review examines conceptual, functional, and molecular features of the first-generation phage display vector pComb3 and its successors, pComb3H, pComb3X, and pC3C.
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