Phage display: applications, innovations, and issues in phage and host biology

Wilson, D.R.; Finlay, B. Brett

doi:10.1139/cjm-44-4-313

Cited by 44 publications

(19 citation statements)

References 164 publications

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“…Stretches of positively-charged amino acids are known to serve as a secretion block, preventing protein translocation across the cytosolic membrane (Wilson and Finlay, 1998). To circumvent this problem, we expressed highly-positive constructs in the cytosol using a vector (pET21a) that lacks the pelB signal peptide.…”

Section: Resultsmentioning

confidence: 99%

Structure-Based Design of Supercharged, Highly Thermoresistant Antibodies

Miklos

Kluwe

Der

et al. 2012

Chemistry & Biology

128

133

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SUMMARY Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that “supercharges” proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ≈30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.

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Section: Resultsmentioning

confidence: 99%

Structure-Based Design of Supercharged, Highly Thermoresistant Antibodies

Miklos

Kluwe

Der

et al. 2012

Chemistry & Biology

128

133

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“…(iii) Measure all copy numbers of all sequences, including zero values, with high confidence. Requirement (i) has been an ongoing effort in our group [11, 40] and other groups [13, 41–43]; for review see [11, 44]. Deep sequencing makes it simple to satisfy requirement (ii) and obtain multiple instances of the same experiment.…”

Section: Theoretical Descriptionmentioning

confidence: 99%

Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

Matochko

Derda

2013

Computational and Mathematical Methods in Medicine

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Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N × 1 frequency vector n = ||ni||, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N × N matrix and a stochastic sampling operator (S a). The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of S a and use them to define the sequencing operator (S e q). Sequencing without any bias and errors is S e q = S a IN, where IN is a N × N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (C E N), which describes elimination or statistically significant downsampling, of specific reads during the sequencing process.

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“…Makowski and coworkers quantified bias in pIII-displayed libraries (22,32), and used this information to develop a predictor of sequence-specific censorship (33). Periplasmic export of phage proteins through the Sec pathway, in general, was found to be a detriment to the display of globular proteins; this bias could be overcome by switching from Sec to other export pathways (34) [for an in-depth review of mechanistic origin of bias see (35)]. Finally, sequence-independent bias caused by mutations in regulatory regions of the phage genome has been uncovered by research groups of Smith and Noren (36,37).…”

Section: Introductionmentioning

confidence: 99%

Prospective identification of parasitic sequences in phage display screens

Matochko

Tang

et al. 2013

Nucleic Acids Research

112

132

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Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones.

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Phage display: applications, innovations, and issues in phage and host biology

Cited by 44 publications

References 164 publications

Structure-Based Design of Supercharged, Highly Thermoresistant Antibodies

Structure-Based Design of Supercharged, Highly Thermoresistant Antibodies

Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

Prospective identification of parasitic sequences in phage display screens

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