Modifying Filamentous Phage Capsid: Limits in the Size of the Major Capsid Protein

Iannolo, Gioacchin; Minenkova, Olga; Petruzzelli, Raffaele; Cesareni, Gianni

doi:10.1006/jmbi.1995.0264

Cited by 140 publications

(123 citation statements)

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“…The resin was introduced in the ABI-431A peptide synthesizer and all Fmoc-amino acids were coupled using the DCC/HOBt protocol from the company (4-fold excess of the Fmoc-amino acid, 90 min per coupling). The peptide was cleaved from the resin with a mixture of TFA: TIS: H 2 O (95%: 2.5%: 2.5%) (5 ml) yielding the crude DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala peptide which was precipitated and washed with cold ether (5 ml X 5). The crude DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala (385 mg) was purified by preparative HPLC (gradient b) and lyophilized to give 170 mg of the peptide product as a white solid.…”

Section: Dup-1 (1-12) 2 Ala (Fapnraqdyntn-amide) Peptidementioning

confidence: 99%

“…Cells were placed in 1.5ml tubes. In parallel, unlabeled DUP-1, DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala, 15-mer and pegylated Rak-2 [1] were dissolved in fresh RPMI containing 1% BSA at different concentrations (from 50 nM to 0.6 mM). 250 µl of each concentration were transferred to the 1.5ml tubes containing the cells and pre-incubated for 40 min at 37 0 C in the orbital shaker.…”

Section: -General Procedures For Hit Validation By a Cell Based Flow mentioning

confidence: 99%

“…To date the quantity of ligands that can be tested as mixtures is limited, we are able to introduce 6 levels, thus if one level is used for the positive calibration control KLBM, we can use mixtures of up to 5 MBM (1)(2)(3)(4)(5) at a time. However, it is possible to do better if we prepare combinatorial peptide libraries so that each member is a mixture of several peptides sharing a common sequence except for a specific position where a mixture of amino acids is applied, thus we can introduce point-mixtures and compare their affinity for the cells assuming that the difference in affinities will be induced by the convoluted position (we call this approach biased inverted positional screening).…”

Section: Combinatorial Peptide Libraries Synthesized On the Phage Likmentioning

confidence: 99%

“…Compound Rak-2, previously validated as having good affinity for PC-3 cells, was synthesized in a pegylated form to improve solubilization and analyzed together with the new peptide candidate. (FITC-13 Lys)DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) standard peptide was incubated with PC-3 cells at a concentration of 1µM in the presence of different concentrations of non-labeled DUP-1, negative control peptide 15-mer or ligand hits as previously shown [1]. The cells were analyzed by flowcytometry and the affinity was determined using the geometric means obtained from FACS plots at the different concentrations of ligands [13].…”

Section: Validation Studiesmentioning

confidence: 99%

“…After incubation and washing procedures similar to those used in phage display peptide *Address correspondence to this author at the Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel; Fax: +972-3-7384053; Tel: +972 3 5318325; E-mail: bykger@mail.biu.ac.il screening, bound small molecules are detected by flow cytometry based on the differential fluorescence intensities of the different microspheres. A major interest of our new synthetic phage-like system is to screen small molecules, unnatural peptides and peptides longer than 12 amino acids that cannot be expressed in phage systems for screening [2][3][4]. In a first proof of concept study [1] we have assessed the system for a library of small molecules obtained by the known Ugi reaction.…”

Section: Introductionmentioning

confidence: 99%

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A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

Khandadash¹,

Partouche²,

Weiss³

et al. 2011

TOOPTSJ

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Herein we present a new application of a recently demonstrated fully synthetic "phage-like" system for screening of combinatorial mixtures in a live cell assay. The new application includes the direct synthesis of peptides and combinatorial libraries on 2 µm cross-linked mono-dispersed microspheres bearing a panel of fluorescence tags. Their characterization using classical chemical analysis as well as biological recognition of the synthesized sequences on the microspheres by specific antibodies, demonstrate the robustness of the system. Two biased positional combinatorial peptide libraries derived from peptide DUP-1 were synthesized and screened for affinity to prostate cancer PC-3 cell line as compared to DUP-1, a peptide with good affinity for this cell line. The best sub-library was deconvoluted and mixtures of deconvoluted peptides on microspheres were screened for identifying the sequences with the best affinity for cells. The best peptide, DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala with high affinity for PC-3 cells, was individually synthesized and validated in an independent binding assay. Finally, the cellular fate of DUP-1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) 2 Ala in PC-3 cell line is demonstrated using confocal laser scanning microscopy. Overall, these results demonstrate that the synthetic phage-like system recently assessed for screening mixtures of small organic molecules, can be also used for synthesis and screening of combinatorial libraries of peptides in live cell assays.

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Section: Dup-1 (1-12) 2 Ala (Fapnraqdyntn-amide) Peptidementioning

confidence: 99%

Section: -General Procedures For Hit Validation By a Cell Based Flow mentioning

confidence: 99%

Section: Combinatorial Peptide Libraries Synthesized On the Phage Likmentioning

confidence: 99%

Section: Validation Studiesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

Khandadash¹,

Partouche²,

Weiss³

et al. 2011

TOOPTSJ

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Shuffling of structural elements in filamentous bacteriophages

Kishchenko

Makowski

1997

Proteins

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All class II filamentous bacteriophage coat proteins contain a conserved, 12-amino acid sequence highly homologous to the loop portion of the EF-hand Ca(2+)-binding motif. The Pf3 coat protein contains two regions of homology to this sequence. The 12-amino acid sequence corresponds to a region of the Pf1 coat protein whose structure is controversial. In some models of the virus structure, this region is alpha-helical. In others, it forms a loop that folds back on itself. The similarity of this region to the loop in the helix-loop-helix Ca(2+)-binding motif suggests that it takes on a loop structure in the virion. Each filamentous phage lacks at least one residue normally involved in Ca(2+)-coordination, consistent with the relatively weak Ca(2+)-binding properties of the filamentous phages. Consideration of the structure of the coat protein in the membrane and in the virus particle indicates that the protein may be more effective in binding cations in its membrane-bound form than in the virus particle. This suggests that release of cations from this loop may be an obligate step during assembly of the proteins into the virus particle.

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The Use of Phage Display in Neurobiology

Bradbury

1999

CP Neuroscience

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Phage display is a technique that involves the coupling of phenotype to genotype in a selectable format. It has been extensively used in molecular biology to study protein-protein interactions, receptor and antibody binding sites, and immune responses; to modify protein properties; and to select antibodies against a wide range of different antigens. In the format most often used, a polypeptide is displayed on the surface of a filamentous phage by genetic fusion to one of the coat proteins, creating a chimeric coat protein. As the gene encoding the chimeric coat protein is packaged within the phage, selection of the phage on the basis of the binding properties of the polypeptide displayed on the surface simultaneously results in the isolation of the gene encoding the polypeptide. This unit describes the background of the technique and illustrates how it has been applied to a number of different problems, each of which has its neurobiological counterparts.

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Modifying Filamentous Phage Capsid: Limits in the Size of the Major Capsid Protein

Cited by 140 publications

References 0 publications

A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

A Fully Synthetic “Phage-Like” System II: Synthesis and Live Cell Screening of Combinatorial Libraries of Peptides on Sub-Cellular Sized Microspheres

Shuffling of structural elements in filamentous bacteriophages

The Use of Phage Display in Neurobiology

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