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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Related Experiment Video

Updated: Mar 6, 2026

Long-range Channelrhodopsin-assisted Circuit Mapping of Inferior Colliculus Neurons with Blue and Red-shifted Channelrhodopsins
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Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins.

Claire N Bedbrook1, Austin J Rice2, Kevin K Yang2

  • 1Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.

Proceedings of the National Academy of Sciences of the United States of America
|March 12, 2017
PubMed
Summary

Structure-guided SCHEMA recombination created diverse channelrhodopsin (ChR) chimeras. Many chimeras improved membrane localization and function, offering insights into integral membrane protein engineering.

Keywords:
channelrhodopsinchimeragenesismembrane proteinsstructure-guided recombination

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Protein Engineering

Background:

  • Integral membrane proteins (MPs) are crucial for cell function and are key targets for engineering.
  • Modifying MPs often presents challenges in maintaining membrane localization while altering other properties.

Purpose of the Study:

  • To engineer diverse channelrhodopsin (ChR) chimeras using structure-guided SCHEMA recombination.
  • To assess the expression, plasma membrane localization, and function of these ChR chimeras.

Main Methods:

  • Utilized structure-guided SCHEMA recombination to generate chimeras from three sequence-diverse ChRs.
  • Assayed 218 ChR chimeras for expression and plasma membrane localization in human embryonic kidney cells.
  • Evaluated functionality, light-activated currents, and kinetic/spectral properties of successful chimeras.

Main Results:

  • The majority of generated chimeras demonstrated improved expression compared to parent ChRs (89% outperformed the lowest-expressing parent).
  • A significant portion (23%) exhibited enhanced plasma membrane localization.
  • Most well-localizing chimeras (93%) retained functionality as light-gated channels, with some showing superior currents and unique properties.

Conclusions:

  • SCHEMA recombination is an effective method for generating protein sequence and functional diversity in integral membrane proteins.
  • This approach provides valuable insights into sequence-function relationships within membrane proteins.
  • Engineered ChR chimeras can exhibit improved membrane localization and novel functional characteristics.