Ligand-Mediated Protection against Phage Lysis as a Positive Selection Strategy for the Enrichment of Epitopes Displayed on the Surface of E. coli Cells
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Peter Camaj
Abstract
We present a novel strategy, termed CISTEM, which allows direct in vivo screening of polypeptides displayed on the surface of E. coli cells by a combination of ligandmediated protection and phagemediated selection. The effectiveness of this new approach was demonstrated by displaying the T7tag on the surface of E. coli as a fusion with the outer membrane protein A, the receptor for bacteriophage K3. A monoclonal T7tag antibody was used as protective ligand for T7tagdisplaying cells and phage K3 for the elimination of unprotected cells. When populations of bacteria, containing between 6 to 10 000 cells displaying the T7tag and approximately 10exp8 cells displaying an unrelated OmpA fusion protein, were infected with phage K3, specific and antibodydependent survival of T7tag displaying cells was observed, yielding an enrichment factor of up to 10exp7-fold. The CISTEM technology was used to select sequences from a T7tagbased, randomised library and the results were compared to those obtained from selection by MACS with the same library. Together, these results reveal a novel in vivo screening strategy in which an E. coli phage receptor is used as display platform and selection is performed in suspension upon addition of a protective ligand and a bacteriophage. Extentions and modifications of the basic strategy should lead to novel applications for the identification of proteinligand interactions.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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- Ligand-Mediated Protection against Phage Lysis as a Positive Selection Strategy for the Enrichment of Epitopes Displayed on the Surface of E. coli Cells
- Structural and Redox Properties of the Leaderless DsbE (CcmG) Protein: Both Active-Site Cysteines of the Reduced Form Are Involved in Its Function in the Escherichia coli Periplasm
- Polyphenols of Cocoa: Inhibition of Mammalian 15-Lipoxygenase
- Total Antioxidant Capacity and Nuclear DNA Damage in Keratinocytes after Exposure to H2 O2
- Recombinant Cryptic Human Fibronectinase Cleaves Actin and Myosin: Substrate Specificity and Possible Role in Muscular Dystrophy
- Rat Tripeptidyl Peptidase I: Molecular Cloning, Functional Expression, Tissue Localization and Enzymatic Characterization
- Determination of NADH in Frozen Rat Brain Sections by Laser-Induced Fluorescence
- Human -Calpain: Simple Isolation from Erythrocytes and Characterization of Autolysis Fragments
- Erratum
- Acknowledgement
- Content Index
- Author Index
- Subject Index
Articles in the same Issue
- New Control of Mitochondrial Membrane Potential and ROS Formation A Hypothesis
- Brix from Xenopus laevis and Brx1p From Yeast Define a New Family of Proteins Involved in the Biogenesis of Large Ribosomal Subunits
- Mig-6 Is a Negative Regulator of the Epidermal Growth Factor Receptor Signal
- β-Carotene Inhibits Growth of Human Colon Carcinoma Cells in Vitro by Induction of Apoptosis
- Ligand-Mediated Protection against Phage Lysis as a Positive Selection Strategy for the Enrichment of Epitopes Displayed on the Surface of E. coli Cells
- Structural and Redox Properties of the Leaderless DsbE (CcmG) Protein: Both Active-Site Cysteines of the Reduced Form Are Involved in Its Function in the Escherichia coli Periplasm
- Polyphenols of Cocoa: Inhibition of Mammalian 15-Lipoxygenase
- Total Antioxidant Capacity and Nuclear DNA Damage in Keratinocytes after Exposure to H2 O2
- Recombinant Cryptic Human Fibronectinase Cleaves Actin and Myosin: Substrate Specificity and Possible Role in Muscular Dystrophy
- Rat Tripeptidyl Peptidase I: Molecular Cloning, Functional Expression, Tissue Localization and Enzymatic Characterization
- Determination of NADH in Frozen Rat Brain Sections by Laser-Induced Fluorescence
- Human -Calpain: Simple Isolation from Erythrocytes and Characterization of Autolysis Fragments
- Erratum
- Acknowledgement
- Content Index
- Author Index
- Subject Index
