Molecular Biotechnology

, Volume 54, Issue 3, pp 829–841 | Cite as

Directed Evolution of a Fluorogen-Activating Single Chain Antibody for Function and Enhanced Brightness in the Cytoplasm

  • Bradley P. Yates
  • Michelle A. Peck
  • Peter B. Berget
Research

Abstract

Directed evolution is an exceptionally powerful tool that uses random mutant library generation and screening techniques to engineer or optimize functions of proteins. One class of proteins for which this process is particularly effective is antibodies, where properties such as antigen specificity and affinity can be selected to yield molecules with improved efficacy as molecular labels or in potential therapeutics. Typical antibody structure includes disulfide bonds that are required for stability and proper folding of the domains. However, these bonds are unable to form in the reducing environment of the cytoplasm, stymieing the effectiveness of optimized antibodies in many research applications. We have removed disulfide-forming cysteine residues in a single chain antibody fluorogen-activating protein (FAP), HL4, and employed directed evolution to select a derivative that is capable of activity in the cytoplasm. A subsequent round of directed evolution was targeted at increasing the overall brightness of the fluoromodule (FAP–fluorogen complex). Ultimately, this approach produced a novel FAP that exhibits strong activation of its cognate fluorogen in the reducing environment of the cytoplasm, significantly expanding the range of applications for which fluoromodule technology can be utilized.

Keywords

Directed evolution Fluorogen activating protein Fluoromodule Intrabody scFv Fluorogenic dyes 

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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Bradley P. Yates
    • 1
  • Michelle A. Peck
    • 1
  • Peter B. Berget
    • 1
    • 2
  1. 1.Department of Biological Sciences & Molecular Biosensor and Imaging CenterCarnegie Mellon UniversityPittsburghUSA
  2. 2.Department of Biological SciencesThe University of the SciencesPhiladelphiaUSA

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