Construction of High-Complexity Combinatorial Phage Display Peptide Libraries

Noren, Karen A.; Noren, Christopher J.

doi:10.1006/meth.2000.1118

Cited by 118 publications

(89 citation statements)

References 46 publications

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“…M13KE vectors with stop codon in the N-terminal region of the pIII gene would lack N-terminal leader sequence and would not produce viable phage. [26] We concluded that TGA codons and other non-NNK codons are sequencing errors.…”

Section: Overview Of the Scriptsmentioning

confidence: 85%

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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: Overview Of the Scriptsmentioning

confidence: 85%

“…We attempted to isolate the library sequences using KpnI and EagI restriction enzymes to isolate variable domains from M13KE vectors [26]. The collection of sticky-end fragments could be repaired to give blunt-ended fragments with identical termini.…”

Section: Isolation Of Variable Ds Dna Fragments From Phage Librariesmentioning

confidence: 99%

Deep sequencing analysis of phage libraries using Illumina platform

Matochko

Chu

Jin

et al. 2012

Methods

110

114

View full text Add to dashboard Cite

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“…[14] The 167 amino acid long cellulose-binding domain (CBD) from Clostridium thermocellum served as the infectivity-reducing tag, [15] as fusion proteins with > 100 amino acids are known to reduce the infectivity of pIII. [16] Additionally, it had been shown previously that this tag is resistant against a viral serine protease. [17] For verification, phages containing a sensitive (AAFAAF) and a more resistant (KHDKRD) hexapeptide substrate were initially generated.…”

Section: Resultsmentioning

confidence: 99%

Modulation of Infectivity in Phage Display as a Tool to Determine the Substrate Specificity of Proteases

et al. 2006

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Proteases play an important role in human and animal diseases. Rapid determination of substrate specificity is possible through the use of substrate phage display; however, current methods possess several drawbacks. They require phage-immobilization and cannot be used for infectivity-destroying or affinity tag-destroying proteases; this can make entire libraries useless. To overcome these limitations, here we introduce infectivity-modulated phage display (IMOP). IMOP uses a protease-resistant and infectivity-reducing tag fused to substrate-displaying polyvalent phages, and the specific cleavage of the substrate increases the infectivity of the phages by releasing the infectivity-reducing tag. The resulting phages were first tested with the infectivity-destroying detergent protease subtilisin; this resulted in a highly specific substrate at a 200-fold enrichment. In a second example, the protease ompT was used and led to an enrichment of the known double-arginine motif. The IMOP system thus substantially improves and simplifies previous systems.

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“…1,[3][4][5] In this technology, highly diverse libraries can be constructed allowing rapid identification and isolation of specific ligands for numerous protein targets such as enzymes, and cell surface receptors. 5,6 Structural analysis of the selected ligands bound to their targets could provide detailed information regarding ligand-target interaction which can be useful in drug discovery and development. Recent advances in phage display technology and antibody engineering have paved the way for the development of phage-displayed antibody technology.…”

Section: Introductionmentioning

confidence: 99%