Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors

Roberts, Bruce; Markland, William; Siranosian, Kathryn Jaacks; Saxena, M.; Guterman, Sonia K.; Ladner, Robert C.

doi:10.1016/0378-1119(92)90156-j

Cited by 46 publications

(13 citation statements)

References 21 publications

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“…Bacteriophage display permits the expression of millions of peptides (14,15) or proteins (16)(17)(18) on the surface of bacteriophage particles, biochemical selection of ligands, and identification by DNA sequencing of the packaged bacteriophage genomes. Peptide ligands for several soluble proteins including streptavidin, concanavalin A, integrins, such as gpllb,, and a variety of antibodies have been identified (15,(19)(20)(21)(22) as well as antibody fiagments that bind to cells (23).…”

mentioning

confidence: 99%

High-affinity urokinase receptor antagonists identified with bacteriophage peptide display.

Goodson¹,

Doyle²,

Kaufman³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

178

129

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The migration and invasion of cells are necessary for many normal and pathological processes, including tissue remodeling, embryo implantation, angiogenesis, and tumor cell invasion and metastasis (1-4). Recent reports suggest that these processes require an active cell-surface proteolytic cascade (5, 6). Important components of this cascade are the plasminogen activator/plasmin system, as well as the matrix metalloproteinases (6). The requirement for both protease expression and a cell-surface protease binding protein has been demonstrated most clearly in the case of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR) but has also been recently described for type IV collagenase (7,8). It has been shown in human colon and breast carcinomas that urokinase is expressed in stromal, fibroblast-like cells and uPAR is expressed on tumor epithelial cells or macrophages, respectively (9, 10). This suggests that a paracrine relationship between uPA and its receptor occurs in these pathological conditions. The in vitro observation that human tumor cell invasion is proportional to receptor-bound urokinase, not total urokinase synthesis, further supports the hypothesis that cell-surface protease localization is a key for invasion (11,12). Other results show that plasminogen activation is more efficient when both uPA and plasminogen are bound on a cell surface, and that cell-surface plasmin is resistant to inhibition by a2-antiplasmin (7 (23). We report here the identification and characterization of peptide antagonists with nanomolar affinity for the human uPAR by using a 15-mer peptide library. This extension of bacteriophage peptide display to cell-surfaceexpressed proteins expands the utility of the method to a wide variety of biologically interesting targets. MATERIALS AND METHODSReagets and Stains. Bacteriophage library construction and bacteriophage growth and isolation were performed as described by Devlin et al. (15). The Escherichia coli strains H249, a recA, sup0, F' derivative of MM294, and JM103 [F' traD36 proAB+ lacJq lacZAM15 A(pro-lac) supE hsdR endAI sbcBl5 thi-1 strAAi] were used for these experiments. Recombinant DNA manipulations were according to Sambrook et aL (24); electrocompetent E. coil HB101 (Stratagene) were used for subcloning unless otherwise noted. Restriction enzymes were from New England Biolabs; high molecularweight human uPA, plasminogen, and the anti-uPAR monoclonal antibody 3936 were from American Diagnostica (Greenwich, CT). Streptavidin was from Molecular Probes or Sigma, and bovine serum albumin (BSA) was from Sigma. Immulon-2 96-well plates were fom Dynatech. The plasmin substrate S-2251 was from Kabi Pharmacia Diagnostics (Piscataway, NJ). Linear synthetic peptides were prepared on an Applied Biosystems model 430A peptide synthesizer using 9-fluorenylmethoxycarbonyl-based chemistry and were purified by reversed-phase HPLC after trifluoroacetic acid cleavage. Alternatively, peptides were obtained from Chiron Mimotopes (Melbourne, Australia). The cyclic uPA peptide encom...

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mentioning

confidence: 99%

High-affinity urokinase receptor antagonists identified with bacteriophage peptide display.

Goodson¹,

Doyle²,

Kaufman³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

178

129

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“…Elution of the specifically bound antibodies is performed by using acid solutions, such as HCl or a glycine buffer [78, 82], or basic (alkaline) solutions, such as triethylamine [30]. It is very important to neutralize the phage antibody eluate right after elution, adjusting the pH to 7.2-7.4.…”

Section: Affinity Selection Of Antibodies From Librariesmentioning

confidence: 99%

Phage Display on the Base of Filamentous Bacteriophages: Application for Recombinant Antibodies Selection

Tikunova

Morozova

2009

Acta Naturae

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The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

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“…DX-890 (Dyax Corp., USA) is a potent and selective smallprotein inhibitor of NE which was discovered by phage display [14]. This study investigated if DX-890 could reduce NE activity from activated neutrophils and IL-8 secretion from airway epithelial cells, and if DX-890 could reduce neutrophil trans-epithelial migration.…”

Section: Introductionmentioning

confidence: 99%

Anti-inflammatory effects of DX-890, a human neutrophil elastase inhibitor

Dunlevy

Martin

Courcey

et al. 2012

Journal of Cystic Fibrosis

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Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors

Cited by 46 publications

References 21 publications

High-affinity urokinase receptor antagonists identified with bacteriophage peptide display.

High-affinity urokinase receptor antagonists identified with bacteriophage peptide display.

Phage Display on the Base of Filamentous Bacteriophages: Application for Recombinant Antibodies Selection

Anti-inflammatory effects of DX-890, a human neutrophil elastase inhibitor

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