Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 106, Issue 29, Pages 11895-11900Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0902828106
Keywords
alternative scaffold; directed evolution; yeast surface display; thermal stability; loop randomization
Categories
Funding
- National Institutes of Health [NS052649, 5T32HG002760]
- University of Wisconsin Graduate School
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Proteins that can bind specifically to targets that also have an intrinsic property allowing for easy detection could facilitate a multitude of applications. While the widely used green fluorescent protein (GFP) allows for easy detection, attempts to insert multiple binding loops into GFP to impart affinity for a specific target have been met with limited success because of the structural sensitivity of the GFP chromophore. In this study, directed evolution using a surrogate loop approach and yeast surface display yielded a family of GFP scaffolds capable of accommodating 2 proximal, randomized binding loops. The library of potential GFP-based binders or GFAbs was subsequently mined for GFAbs capable of binding to protein targets. Identified GFAbs bound with nanomolar affinity and required binding contributions from both loops indicating the advantage of a dual loop GFAb platform. Finally, GFAbs were solubly produced and used as fluorescence detection reagents to demonstrate their utility.
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