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Updated: Jul 5, 2026

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Published on: February 16, 2014

Using T7 phage display to select GFP-based binders.

M Dai¹, J Temirov, E Pesavento

¹Biosciences Division, Los Alamos National Laboratory, Los Alamos, NM, USA.

Protein Engineering, Design & Selection : PEDS

|May 13, 2008

Summary

This summary is machine-generated.

T7 phage display effectively presents cytoplasmic proteins like green fluorescent protein (GFP). Selected GFP-based binders from T7 libraries enable sensitive detection without secondary reagents.

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Area of Science:

Biotechnology
Molecular Biology
Protein Engineering

Background:

Traditional phage display is inefficient for cytoplasmic proteins.
Filamentous phage have limitations in displaying intracellular proteins effectively.

Purpose of the Study:

To evaluate T7 phage as a platform for displaying cytoplasmic proteins, specifically green fluorescent protein (GFP) variants.
To develop and select specific GFP-based affinity reagents using T7 phage display for sensitive detection applications.

Main Methods:

Utilized T7 phage display to present GFP derivatives with binding loops.
Employed single-molecule detection techniques (fluorescence correlation spectroscopy, anti-bunching) to quantify displayed proteins.
Optimized selection conditions using mixtures of binders and non-binders.
Screened a library of T7-displayed GFP clones with CDR3 antibody binding loops.

Main Results:

Demonstrated successful display of 1-3 GFP molecules per T7 phage particle.
Optimized selection strategies for isolating specific binders.
Selected GFP-based binders capable of target recognition.
Validated the use of selected binders in flow cytometry, fluorescence-linked immunosorbant assays, and immunoblotting without secondary reagents.

Conclusions:

T7 phage display is an effective system for presenting cytoplasmic proteins like GFP.
Selected GFP-based affinity reagents offer intrinsic fluorescence detection capabilities.
This approach enables sensitive detection in various biological assays, reducing the need for secondary labeling.