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      PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries

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          Abstract

          The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE ( Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized ‘expected’ occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

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          Most cited references23

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          Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface.

          G. Smith (1985)
          Foreign DNA fragments can be inserted into filamentous phage gene III to create a fusion protein with the foreign sequence in the middle. The fusion protein is incorporated into the virion, which retains infectivity and displays the foreign amino acids in immunologically accessible form. These "fusion phage" can be enriched more than 1000-fold over ordinary phage by affinity for antibody directed against the foreign sequence. Fusion phage may provide a simple way of cloning a gene when an antibody against the product of that gene is available.
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            Filamentous phage display in the new millennium.

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              Teaching an old scaffold new tricks: monobodies constructed using alternative surfaces of the FN3 scaffold.

              The fibronectin type III domain (FN3) has become one of the most widely used non-antibody scaffolds for generating new binding proteins. Because of its structural homology to the immunoglobulin domain, combinatorial libraries of FN3 designed to date have primarily focused on introducing amino acid diversity into three loops that are equivalent to antibody complementarity-determining regions. Here, we report an FN3 library that utilizes alternative positions for presenting amino acid diversity. We diversified positions on a β-sheet and surface loops that together form a concave surface. The new library produced binding proteins (termed "monobodies") to multiple target proteins, generally with similar efficacy as the original, loop-focused library. The crystal structure of a monobody generated from the new library in complex with its target, the Abl SH2 domain, revealed that a concave surface of the monobody, as intended in our design, bound to a convex surface of the target with the interface area being among the largest of published structures of monobody-target complexes. This mode of interaction differs from a common binding mode for single-domain antibodies and antibody mimics in which recognition loops recognize clefts in targets. Together, this work illustrates the utilization of different surfaces of a single immunoglobulin-like scaffold to generate binding proteins with distinct characteristics. Copyright © 2011 Elsevier Ltd. All rights reserved.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: Writing – original draft
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: MethodologyRole: Writing – review & editing
                Role: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                23 February 2018
                2018
                : 13
                : 2
                : e0193332
                Affiliations
                [001]School of Biological Sciences and Edinburgh Medical School: Biomedical Sciences, University of Edinburgh, The King’s Buildings, Edinburgh, Scotland, United Kingdom
                AC Camargo Cancer Hospital, BRAZIL
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0001-8920-3522
                Article
                PONE-D-17-37342
                10.1371/journal.pone.0193332
                5825087
                29474422
                3cf67878-a527-4d02-b25e-9d32572dff4e
                © 2018 Shave et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 18 October 2017
                : 8 February 2018
                Page count
                Figures: 3, Tables: 0, Pages: 10
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000265, Medical Research Council;
                Award ID: J54359
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100011260, FP7 Research infrastructures;
                Award ID: 278568
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 203149
                Award Recipient :
                Funded by: Scottish Universities Life Sciences Alliance
                Award Recipient :
                The authors acknowledge financial support from the Scottish Universities Life Sciences Alliance ((SULSA- http://www.sulsa.ac.uk) (MA, SS, SM, JK), the Medical Research Council ((MRC- www.mrc.ac.uk, J54359) Strategic Grant (MA), and from the European Community’s 7th Framework Program (FP7/2007–2013) under grant agreement no 278568 ‘PRIMES’ (MA, SS). The Wellcome Trust Centre for Cell Biology is supported by core funding from the Wellcome Trust [203149] (AK).
                Categories
                Research Article
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Molecular Biology Display Techniques
                Phage Display
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Molecular Biology Display Techniques
                Phage Display
                Biology and life sciences
                Genetics
                DNA
                DNA libraries
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA libraries
                Biology and Life Sciences
                Biochemistry
                Proteomics
                Peptide Libraries
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Next-Generation Sequencing
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Next-Generation Sequencing
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Transcriptome Analysis
                Next-Generation Sequencing
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Transcriptome Analysis
                Next-Generation Sequencing
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Amino Acid Sequence Analysis
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Motif Analysis
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Library Screening
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Library Screening
                Research and analysis methods
                Database and informatics methods
                Bioinformatics
                Sequence analysis
                DNA sequence analysis
                Custom metadata
                The PuLSE software (source and binaries) along with an example dataset is available at: https://github.com/stevenshave/PuLSE

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                Uncategorized

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