Peptide Phage Display for Discovery of Novel Biomarkers for Imaging and Therapy of Cell Subpopulations in Ovarian Cancer

Soendergaard, Mette; Northup, Jessica R Newton-

doi:10.4172/2155-9929.s2-004

Cited by 5 publications

(7 citation statements)

References 84 publications

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“…The traditional screening approaches, which are based on agar plate or microplate assays, would require robotics to handle such large libraries. In the absence of robust screening methods, it seems difficult or even impossible to identify enzyme variants with desired properties. − If screening approaches are not amenable to automation, only about 1000 to 2000 of the mutants can be screened for desired properties. , Cell surface display combined with target-displaying magnetic beads for panning rounds has been widely used in antibody , and drug , discovery. In this study, a lanthipeptide L. lactis display system was applied .…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB

Zhao

Cebrián

et al. 2020

ACS Synth. Biol.

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Microbial lanthipeptides are formed by a two-step enzymatic introduction of (methyl)lanthionine rings. A dehydratase catalyzes the dehydration of serine and threonine residues, yielding dehydroalanine and dehydrobutyrine, respectively. Cyclase-catalyzed coupling of the formed dehydroresidues to cysteines forms (methyl)lanthionine rings in a peptide. Lanthipeptide biosynthetic systems allow discovery of target-specific, lanthionine-stabilized therapeutic peptides. However, the substrate specificity of existing modification enzymes impose limitations on installing lanthionines in non-natural substrates. The goal of the present study was to obtain a lanthipeptide dehydratase with the capacity to dehydrate substrates that are unsuitable for the nisin dehydratase NisB. We report high-throughput screening for tailored specificity of intracellular, genetically encoded NisB dehydratases. The principle is based on the screening of bacterially displayed lanthionine-constrained streptavidin ligands, which have a much higher affinity for streptavidin than linear ligands. The designed NisC-cyclizable high-affinity ligands can be formed via mutant NisB-catalyzed dehydration but less effectively via wild-type NisB activity. In Lactococcus lactis, a cell surface display precursor was designed comprising DSHPQFC. The Asp residue preceding the serine in this sequence disfavors its dehydration by wild-type NisB. The cell surface display vector was coexpressed with a mutant NisB library and NisTC. Subsequently, mutant NisBcontaining bacteria that display cyclized strep ligands on the cell surface were selected via panning rounds with streptavidin-coupled magnetic beads. In this way, a NisB variant with a tailored capacity of dehydration was obtained, which was further evaluated with respect to its capacity to dehydrate nisin mutants. These results demonstrate a powerful method for selecting lanthipeptide modification enzymes with adapted substrate specificity.

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Section: Discussionmentioning

confidence: 99%

High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB

Zhao

Cebrián

et al. 2020

ACS Synth. Biol.

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“…on the surface of phage (Reynolds et al, 2011; Soendergaard et al, 2011). The non-lytic M13 filamentous phage is a single-stranded (ss) DNA virus that infects a number of gram-negative bacteria.…”

Section: Introductionmentioning

confidence: 99%

“…Phage display (PD) has taken its stand in the era of molecular biology in the last two decades by displaying a large number of proteins, antibodies, amino acids, etc. on the surface of phage (Reynolds et al, 2011 ; Soendergaard et al, 2011 ). The non-lytic M13 filamentous phage is a single-stranded (ss) DNA virus that infects a number of gram-negative bacteria.…”

Section: Introductionmentioning

confidence: 99%

Construction of helper plasmid-mediated dual-display phage for autoantibody screening in serum

Rajaram

Vermeeren

Somers³

et al. 2014

Appl Microbiol Biotechnol

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M13 filamentous bacteriophage has been used in displaying disease-specific antibodies, biomarkers, and peptides. One of the major drawbacks of using phage in diagnostic assays is the aspecific adsorption of proteins leading to a high background signal and decreasing sensitivity. To deal with this, we developed a genetically pure, exchangeable dual-display phage system in which biomarkers and streptavidin-binding protein (SBP) are displayed at opposite ends of the phage. This approach allows for sample purification, using streptavidin-coated magnetic beads resulting in a higher sensitivity of signal detection assays. Our dual-display cassette system approach also allows for easy exchange of both the anchor protein (SBP) and the displayed biomarker. The presented principle is applied for the detection of antibody reactivity against UH-RA.21 which is a good candidate biomarker for rheumatoid arthritis (RA). The applicability of dual-display phage preparation using a helper plasmid system is demonstrated, and its increased sensitivity in phage ELISA assays using patient serum samples is shown.

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“…Phage-displayed peptides can be used to deliver cargo or to perform functions such as homing to bone marrow and binding to primitive hematopoietic stem cells7, supporting EC/ES cell cultures5, labeling mouse and rhesus monkey ES cells89 and discovering new membrane receptors10. These peptides can also be used as lead compounds in drug design and as alternatives to antibodies for target validation in genomics and drug discovery both in vitro and in vivo 1112. There are several advantages of this bacteriophage amplification system: 1) phage clones display sequences identical to specific peptides; 2) phage clones are simple to prepare and purify; 3) phage recombination can produce induced pluripotent stem cells (iPSCs) with intact endogenous gene function13; and 4) phages can tolerate drastic modifications to their coat proteins and serve as a solid support for organic synthesis14.…”

mentioning

confidence: 99%

Targeting human embryonic stem cells with quantum dot-conjugated phages

Zhao

Jin

Yuan

et al. 2013

Sci Rep

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Targeting embryonic stem cells (ESCs) is important for ESC labeling, drug delivery and cell fate control. In this study, we identified twenty-two phage clones that bind specifically to the hESC cell line X-01, which was derived from human blastocysts of Chinese origin. One phage (H178), which displays the sequence VGGEAWSSPTDL, showed higher binding affinity to hESCs than to a monkey ES cell line (RS366.4) and two mouse ES cell lines (R1 and E14). Using quantum dots (QDs) conjugated to the H178 phage, we demonstrate that the phage can specifically bind to hESCs in vitro. Our results suggest a possible interaction between the selected peptide and the stem cell extracellular matrix (ECM). The selection method described here allows rapid and efficient screening of unique phage clones and targeting cells. The phages displaying peptides identified by this study have potential applications for cargo delivery and receptor studies.

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Peptide Phage Display for Discovery of Novel Biomarkers for Imaging and Therapy of Cell Subpopulations in Ovarian Cancer

Cited by 5 publications

References 84 publications

High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB

High-Throughput Screening for Substrate Specificity-Adapted Mutants of the Nisin Dehydratase NisB

Construction of helper plasmid-mediated dual-display phage for autoantibody screening in serum

Targeting human embryonic stem cells with quantum dot-conjugated phages

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