Chemical Posttranslational Modification of Phage-Displayed Peptides

Ng, Simon; Tjhung, Katrina F.; Paschal, Beth M.; Noren, Christopher J.; Derda, Ratmir

doi:10.1007/978-1-4939-2020-4_11

Cited by 17 publications

(12 citation statements)

References 20 publications

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“…Recently, the scope of phage display extended toward the display of polypeptides containing posttranslational modifi cations, including phosphorylation, phosphopantetheinylation ( Yen and Yin, 2007 ) and glycosylation ( Celik et al, 2010 ; Dürr et al, 2010 ; Ng S. et al, 2015 ). As glycosylation is the most frequent posttranslational modification [>50% of total eukaryotic proteins and an increasing number of archaeal and bacterial proteins ( Abu-Qarn et al, 2008 )], modification of displayed proteins has promising future applications.…”

Section: Phage Displaymentioning

confidence: 99%

Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

et al. 2016

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Microbial surface and secreted proteins (the secretome) contain a large number of proteins that interact with other microbes, host and/or environment. These proteins are exported by the coordinated activities of the protein secretion machinery present in the cell. A group of bacteriophage, called filamentous phage, have the ability to hijack bacterial protein secretion machinery in order to amplify and assemble via a secretion-like process. This ability has been harnessed in the use of filamentous phage of Escherichia coli in biotechnology applications, including screening large libraries of variants for binding to “bait” of interest, from tissues in vivo to pure proteins or even inorganic substrates. In this review we discuss the roles of secretome proteins in pathogenic and non-pathogenic bacteria and corresponding secretion pathways. We describe the basics of phage display technology and its variants applied to discovery of bacterial proteins that are implicated in colonization of host tissues and pathogenesis, as well as vaccine candidates through filamentous phage display library screening. Secretome selection aided by next-generation sequence analysis was successfully applied for selective display of the secretome at a microbial community scale, the latter revealing the richness of secretome functions of interest and surprising versatility in filamentous phage display of secretome proteins from large number of Gram-negative as well as Gram-positive bacteria and archaea.

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Section: Phage Displaymentioning

confidence: 99%

Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

et al. 2016

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“…Therefore, we also generated NNK libraries. We built an N-terminally displayed library with the sequence Ser-XXXX, termed NT-SX4 44 . Previously, we hypothesized that cloning libraries within a different location on pIII could bypass the biases associated with decreased efficiency of periplasmic export of charged sequences 45 .…”

Section: Methodsmentioning

confidence: 99%

“…The majority of display platforms operate with libraries of 10 9 or higher diversity, but this level of diversity cannot be effectively covered using currently available sequencing tools. In this report, we produced three types of small tetrapeptide libraries that have a maximum diversity of 10 5 –10 6 , which is easily accessible to routine Illumina sequencing and data processing: (i) an N-terminally displayed library with the sequence Ser-X-Cys-XXX-Cys, where X is any one of 19 trinucleotide codons (“allowed” codons, cysteine excluded), termed NT-TriNuc 43 ; (ii) an N-terminally displayed library encoded by conventional NNK randomization with the sequence Ser-XXXX, termed NT-SX4 44 ; (iii) a library encoded by NNK randomization and expressed between N1 and N2 domains of phage coat protein pIII, termed ID-SX4 45 . We also developed a paired-end processing program tailored for the analysis of phage-displayed libraries.…”

Section: Introductionmentioning

confidence: 99%

Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing

He¹,

Tjhung²,

Bennett³

et al. 2018

Sci Rep

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Understanding the composition of a genetically-encoded (GE) library is instrumental to the success of ligand discovery. In this manuscript, we investigate the bias in GE-libraries of linear, macrocyclic and chemically post-translationally modified (cPTM) tetrapeptides displayed on the M13KE platform, which are produced via trinucleotide cassette synthesis (19 codons) and NNK-randomized codon. Differential enrichment of synthetic DNA {S}, ligated vector {L} (extension and ligation of synthetic DNA into the vector), naïve libraries {N} (transformation of the ligated vector into the bacteria followed by expression of the library for 4.5 hours to yield a “naïve” library), and libraries chemically modified by aldehyde ligation and cysteine macrocyclization {M} characterized by paired-end deep sequencing, detected a significant drop in diversity in {L} → {N}, but only a minor compositional difference in {S} → {L} and {N} → {M}. Libraries expressed at the N-terminus of phage protein pIII censored positively charged amino acids Arg and Lys; libraries expressed between pIII domains N1 and N2 overcame Arg/Lys-censorship but introduced new bias towards Gly and Ser. Interrogation of biases arising from cPTM by aldehyde ligation and cysteine macrocyclization unveiled censorship of sequences with Ser/Phe. Analogous analysis can be used to explore library diversity in new display platforms and optimize cPTM of these libraries.

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“…An advantage of phage display over the other display methods is that it has been well optimized as it is the oldest and most common display technique. However, phage display does not allow for simple incorporation of many post-translational modifications (e.g., protein glycosylation) without chemical manipulation of the phage after amplification [ 98 ]—in contrast to yeast display.…”

Section: Library Selection and Screening Methods For Gbpsmentioning

confidence: 99%

Resources and Methods for Engineering “Designer” Glycan-Binding Proteins

Warkentin

Kwan

2021

Molecules

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This review provides information on available methods for engineering glycan-binding proteins (GBP). Glycans are involved in a variety of physiological functions and are found in all domains of life and viruses. Due to their wide range of functions, GBPs have been developed with diagnostic, therapeutic, and biotechnological applications. The development of GBPs has traditionally been hindered by a lack of available glycan targets and sensitive and selective protein scaffolds; however, recent advances in glycobiology have largely overcome these challenges. Here we provide information on how to approach the design of novel “designer” GBPs, starting from the protein scaffold to the mutagenesis methods, selection, and characterization of the GBPs.

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Chemical Posttranslational Modification of Phage-Displayed Peptides

Cited by 17 publications

References 20 publications

Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing

Resources and Methods for Engineering “Designer” Glycan-Binding Proteins

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