Quantitative Assessment of Peptide Sequence Diversity in M13 Combinatorial Peptide Phage Display Libraries

Rodi, Diane J.; Soares, Alexei S.; Makowski, Lee

doi:10.1016/s0022-2836(02)00844-6

Cited by 116 publications

(140 citation statements)

References 46 publications

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“…Distribution of amino acids in sequences present at copy number <10,000 was similar to those in the overall library. Overall distribution of amino acids in peptides in the library was similar to those observed in earlier reports [17,21,29]. Library had abundant Ser/Thr in all positions.…”

Section: Preliminary Analysis Of Sequence Diversity In the Librarysupporting

confidence: 85%

“…Abundance of Cys was low in all positions. N-terminus exhibited significant preference for some amino acids, presumably due to proteolytic preference of the peptidase, which truncates leader peptide sequences following the displayed peptide [20,21].…”

Section: Preliminary Analysis Of Sequence Diversity In the Librarymentioning

confidence: 99%

“…In this report, we analyzed diversity of amplified libraries using Illumina and observed a collapse of diversity in phage-displayed libraries after a single round of growth in bacteria. The collapse of the 10 6 -scale library to a few hundred abundant sequences would not be visible in small-scale Sanger sequencing [17,20,21]; it could also have been difficult to detect with smaller-throughput 454 Sequencing.…”

Section: Introductionmentioning

confidence: 99%

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Deep sequencing analysis of phage libraries using Illumina platform

Matochko

Chu

Jin

et al. 2012

Methods

110

114

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This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of the 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 variable regions from a M13KE phage vector. 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 2x10 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.

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Section: Preliminary Analysis Of Sequence Diversity In the Librarysupporting

confidence: 85%

Section: Preliminary Analysis Of Sequence Diversity In the Librarymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Deep sequencing analysis of phage libraries using Illumina platform

Matochko

Chu

Jin

et al. 2012

Methods

110

114

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“…We believe that the wt peptide itself was not selected from the library because of the extremely unfavorable g3p display of peptides presenting an R residue in the Nterminal position [25]. A complete censorship for R at this position (Rp1) is explained by its charge and size that would affect viral morphogenesis [26].…”

Section: Discussionmentioning

confidence: 99%

Phage‐displayed libraries of peptide/major histocompatibility complexes

et al. 2004

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Characterizing peptide epitopes targeted by major histocompatibility complex (MHC)-restricted T cells of unknown specificity would have broad implications. In this article we introduce and validate an original phage-displayed library of noncovalent complexes of peptide and MHC (P/MHC). We show that soluble MHC molecules associate with peptides presented by a phage, thereby resulting in the formation of multivalent P/MHC phages. Complex formation is stabilized by the interaction of the soluble partner (MHC) with two components, peptide and g 2-microglobulin, both of which are covalently linked to the phage. As proof of concept, we have used this strategy to express peptide libraries in the context of H-2K b . Using monoclonal antibody 25D (specific for ovalbumin/H-2K b ) as a template to screen the library, we were able to select a variant epitope functionally and structurally related to the wild-type peptide. Interaction studies between monoclonal antibody 25D and cells suggest that the variant peptide has been selected on the basis of a decreased dissociation rate between the peptide/H-2K b complex and its ligand. A weak agonist of the N15 TCR (vesicular stomatitis virus/H-2K b -specific) was also isolated from another P/MHC library. This strategy opens up new perspectives for antigen discovery and the manipulation of T cell responses.

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“…Filamentous phage (M13, f1, and fd) has been the most common vector used in the construction of peptide phage-displayed libraries [31,33,34], although previous studies demonstrated specific and positional amino acid biases in populations of peptides randomly selected from M13 libraries because of the processes of filamentous phage morphogenesis [35,36]. Alternative phage display systems have been developed using lytic phage such as T7, T4, λ and P4 [37][38][39].…”

Section: Phage-displayed Peptide Library Technologymentioning

confidence: 99%

Potential of phage-displayed peptide library technology to identify functional targeting peptides

Krumpe

Mori²

2007

Expert Opinion on Drug Discovery

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Combinatorial peptide library technology is a valuable resource for drug discovery and development. Several peptide drugs developed through phage-displayed peptide library technology are presently in clinical trials and the authors envision that phage-displayed peptide library technology will assist in the discovery and development of many more. This review attempts to compile and summarize recent literature on targeting peptides developed through peptide library technology, with special emphasis on novel peptides with targeting capacity evaluated in vivo.

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Quantitative Assessment of Peptide Sequence Diversity in M13 Combinatorial Peptide Phage Display Libraries

Cited by 116 publications

References 46 publications

Deep sequencing analysis of phage libraries using Illumina platform

Deep sequencing analysis of phage libraries using Illumina platform

Phage‐displayed libraries of peptide/major histocompatibility complexes

Potential of phage-displayed peptide library technology to identify functional targeting peptides

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