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Cell Chem Biol. 2018 Dec 20;25(12):1554-1561.e8. doi: 10.1016/j.chembiol.2018.08.008. Epub 2018 Sep 13.

Imaging-Based Screening Platform Assists Protein Engineering.

Author information

1
Tools for Bio-Imaging, Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried 82152, Germany.
2
Sensorimotor Control, Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried 82152, Germany.
3
Tools for Bio-Imaging, Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, Martinsried 82152, Germany. Electronic address: griesbeck@neuro.mpg.de.

Abstract

Protein engineering involves generating and screening large numbers of variants for desired properties. While modern DNA technology has made it easy to create protein diversity on the DNA level, the selection and validation of candidate proteins from large libraries remains a challenge. We built a screening platform that integrates high-quality fluorescence-based image analysis and robotic picking of bacterial colonies. It allows tracking each individual colony in a large population and collecting quantitative information on library composition during the protein evolution process. We demonstrate the power of the screening platform by optimizing a dim far-red-emitting fluorescent protein whose brightness increased several fold using iterative cycles of mutagenesis and platform-based screening. The resulting protein variant mCarmine is useful for imaging cells and structures within live tissue as well as for molecular tagging. Overall, the platform presented provides powerful, flexible, and low-cost instrumentation to accelerate many fluorescence-based protein optimization projects.

KEYWORDS:

directed evolution; fluorescent proteins; image analysis; protein engineering; screening

PMID:
30220597
DOI:
10.1016/j.chembiol.2018.08.008
[Indexed for MEDLINE]
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