The Experts below are selected from a list of 2253 Experts worldwide ranked by ideXlab platform
Stephen Dewhurst - One of the best experts on this subject based on the ideXlab platform.
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Display of HIV-1 Envelope Protein on Lambda Phage Scaffold as a Vaccine Platform.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Jonelle L. Mattiacio, Matthew G. Brewer, Stephen DewhurstAbstract:The generation of a strong antibody response to target antigens is a major goal for vaccine development. Here we describe the display of the human immunodeficiency virus (HIV) envelope spike protein (Env) on a virus-like scaffold provided by the lambda Phage capsid. Phage Vectors, in general, have advantages over mammalian virus Vectors due to their genetic tractability, inexpensive production, suitability for scale-up, as well as their physical stability, making them an attractive vaccine platform.
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fc receptor mediated antibody dependent enhancement of bacterioPhage lambda mediated gene transfer in mammalian cells
Virology, 2008Co-Authors: Ramil Sapinoro, Ketna Volcy, W Shanaka W I Rodrigo, Jacob J Schlesinger, Stephen DewhurstAbstract:Abstract Lambda Phage Vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo Phage-mediated gene expression is increased when mice are pre-immunized with bacterioPhage lambda. We now show that, like eukaryotic viruses, bacterioPhage Vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of Phage gene transfer required FcγRI, but not its associated γ-chain, and was not supported by other FcγR family members (FcγRIIA, FcγRIIB, and FcγRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced Phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized Phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced Phage gene transfer. These results reveal an unexpected antibody-dependent, FcγRI-mediated enhancement of Phage transduction in mammalian cells, and suggest new approaches to improve bacterioPhage-mediated gene transfer.
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a tractable method for simultaneous modifications to the head and tail of bacterioPhage lambda and its application to enhancing Phage mediated gene delivery
Nucleic Acids Research, 2007Co-Authors: Christine N Zanghi, Ramil Sapinoro, Birgit Bradeltretheway, Stephen DewhurstAbstract:There is considerable interest in the use of bacterioPhage Vectors for mammalian cell gene transfer applications, due to their stability, excellent safety profile and inexpensive mass production. However, to date, Phage Vectors have been plagued by mediocre performance as gene transfer agents. This may reflect the complexity of the viral infection process in mammalian cells and the need to refine each step of this process in order to arrive at an optimal, Phage-based gene transfer system. Therefore, a flexible system was designed that alowed for the introduction of multiple modifications on the surface of bacterioPhage lambda. Using this novel method, multiple peptides were displayed simultaneously from both the Phage head and tail. Surface head display of an ubiquitinylation motif greatly increased the efficiency of Phage-mediated gene transfer in a murine macroPhage cell line. Gene transfer was further increased when this peptide was displayed in combination with a tail-displayed CD40-binding motif. Overall, this work provides a novel system that can be used to rationally improve bacterioPhage gene transfer Vectors and shows it may be possible to enhance the efficiency of Phage-mediated gene transfer by targeting and optimizing multiple steps within the viral infection pathway.
Ratmir Derda - One of the best experts on this subject based on the ideXlab platform.
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deep sequencing analysis of Phage libraries using illumina platform
Methods, 2012Co-Authors: Wadim L Matochko, George M Whitesides, Ratmir DerdaAbstract:Abstract This paper presents an analysis of Phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacterioPhage (Phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred Phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10 7 reads in a single run. We applied Illumina sequencing to analyze Phage libraries. Using PCR, we isolated the variable regions from M13KE Phage Vectors from a Phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5′-terminal region. We used this approach to examine how diversity of peptides in Phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2 × 10 7 reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6 bp), one complimentary region (12 bp) and a variable region (36 bp). We applied this sequencing to a model library of 10 6 unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in Phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in Phage screens. It could also be instrumental in developing new screening strategies, which can preserve diversity of Phage clones and identify ligands previously lost in Phage display screens.
Ramil Sapinoro - One of the best experts on this subject based on the ideXlab platform.
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fc receptor mediated antibody dependent enhancement of bacterioPhage lambda mediated gene transfer in mammalian cells
Virology, 2008Co-Authors: Ramil Sapinoro, Ketna Volcy, W Shanaka W I Rodrigo, Jacob J Schlesinger, Stephen DewhurstAbstract:Abstract Lambda Phage Vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo Phage-mediated gene expression is increased when mice are pre-immunized with bacterioPhage lambda. We now show that, like eukaryotic viruses, bacterioPhage Vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of Phage gene transfer required FcγRI, but not its associated γ-chain, and was not supported by other FcγR family members (FcγRIIA, FcγRIIB, and FcγRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced Phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized Phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced Phage gene transfer. These results reveal an unexpected antibody-dependent, FcγRI-mediated enhancement of Phage transduction in mammalian cells, and suggest new approaches to improve bacterioPhage-mediated gene transfer.
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a tractable method for simultaneous modifications to the head and tail of bacterioPhage lambda and its application to enhancing Phage mediated gene delivery
Nucleic Acids Research, 2007Co-Authors: Christine N Zanghi, Ramil Sapinoro, Birgit Bradeltretheway, Stephen DewhurstAbstract:There is considerable interest in the use of bacterioPhage Vectors for mammalian cell gene transfer applications, due to their stability, excellent safety profile and inexpensive mass production. However, to date, Phage Vectors have been plagued by mediocre performance as gene transfer agents. This may reflect the complexity of the viral infection process in mammalian cells and the need to refine each step of this process in order to arrive at an optimal, Phage-based gene transfer system. Therefore, a flexible system was designed that alowed for the introduction of multiple modifications on the surface of bacterioPhage lambda. Using this novel method, multiple peptides were displayed simultaneously from both the Phage head and tail. Surface head display of an ubiquitinylation motif greatly increased the efficiency of Phage-mediated gene transfer in a murine macroPhage cell line. Gene transfer was further increased when this peptide was displayed in combination with a tail-displayed CD40-binding motif. Overall, this work provides a novel system that can be used to rationally improve bacterioPhage gene transfer Vectors and shows it may be possible to enhance the efficiency of Phage-mediated gene transfer by targeting and optimizing multiple steps within the viral infection pathway.
Wadim L Matochko - One of the best experts on this subject based on the ideXlab platform.
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deep sequencing analysis of Phage libraries using illumina platform
Methods, 2012Co-Authors: Wadim L Matochko, George M Whitesides, Ratmir DerdaAbstract:Abstract This paper presents an analysis of Phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacterioPhage (Phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred Phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10 7 reads in a single run. We applied Illumina sequencing to analyze Phage libraries. Using PCR, we isolated the variable regions from M13KE Phage Vectors from a Phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5′-terminal region. We used this approach to examine how diversity of peptides in Phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2 × 10 7 reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6 bp), one complimentary region (12 bp) and a variable region (36 bp). We applied this sequencing to a model library of 10 6 unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in Phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in Phage screens. It could also be instrumental in developing new screening strategies, which can preserve diversity of Phage clones and identify ligands previously lost in Phage display screens.
Dario Neri - One of the best experts on this subject based on the ideXlab platform.
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Selection of catalytically active biotin ligase and trypsin mutants by Phage display
Protein Engineering Design and Selection, 2002Co-Authors: Christian Heinis, Julian Bertschinger, Salvatore Demartis, Adrian Huber, Samu Melkko, Luisa Lozzi, Paolo Neri, Dario NeriAbstract:Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on Phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active Phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged Phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by Phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on Phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using Phage Vectors, rather than Phagemid Vectors. In model selection experiments, Phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial Phage display library of trypsin mutants. Selection of catalytically active Phage-enzymes was evidentiated by increasing Phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using Phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.