Genetic Code Expansion of Mammalian Cells with Unnatural Amino Acids

Kalyn A. Brown1, Alexander Deiters1

1 University of Pittsburgh, Department of Chemistry, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch150038
Online Posting Date:  September, 2015
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Abstract

The expansion of the genetic code of mammalian cells enables the incorporation of unnatural amino acids into proteins. This is achieved by adding components to the protein biosynthetic machinery, specifically an engineered aminoacyl‐tRNA synthetase/tRNA pair. The unnatural amino acids are chemically synthesized and supplemented to the growth medium. Using this methodology, fundamental new chemistries can be added to the functional repertoire of the genetic code of mammalian cells. This protocol outlines the steps necessary to incorporate a photocaged lysine into proteins and showcases its application in the optical triggering of protein translocation to the nucleus. © 2015 by John Wiley & Sons, Inc.

Keywords: unnatural amino acid; genetic code expansion; mammalian cell; caging; optogenetics; protein translocation; nuclear localization

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Incorporation of Photocaged Lysine into mCherry‐TAG‐EGFP‐HA
  • Basic Protocol 2: Photochemical Control of Nuclear Localization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Incorporation of Photocaged Lysine into mCherry‐TAG‐EGFP‐HA

  Materials
  • HEK293T cells (ATCC)
  • Complete DMEM with antibiotics (Life Technologies; see recipe)
  • DMEM (see recipe)
  • OptiMEM (Life Technologies)
  • pmCherry‐TAG‐EGFP‐HA (available from Alexander Deiters, University of Pittsburgh or Jason W. Chin, Medical Research Council Laboratory of Molecular Biology; Gautier et al., )
  • pPCKRS/PylT (available from Alexander Deiters, University of Pittsburgh or Jason W. Chin, Medical Research Council Laboratory of Molecular Biology; Gautier et al., )
  • Linear polyethylenimine (L‐PEI) transfection solution (see recipe)
  • Photocaged lysine (PCK) stock solution (100 mM in water; see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Mammalian Protein Extraction buffer (GE Healthcare)
  • 4× SDS loading dye (see recipe)
  • 1× SDS‐PAGE running buffer (see recipe)
  • SDS‐PAGE gel (10%)
  • PVDF membrane (GE Healthcare)
  • 1× Western blot transfer buffer (see recipe)
  • Methanol
  • Anti‐HA antibody (rabbit, Cell Signaling)
  • Anti‐GAPDH antibody (rabbit, Santa Cruz)
  • Powdered milk (Amresco)
  • Tris‐buffered saline with Tween‐20 (TBST; see recipe)
  • Goat anti‐rabbit‐HRP conjugate antibody (Santa Cruz)
  • VisiGlo Prime HRP Chemiluminescent Substrate kit (Amresco)
  • Biological safety cabinet
  • 6‐well plate (Greiner Bio‐One)
  • Incubator (37°C and 5% CO 2)
  • Fluorescence microscope with a 10× objective and filter cubes for mCherry and enhanced‐green fluorescent protein (EGFP) imaging
  • Rocker
  • Refrigerated microcentrifuge
  • Gel apparatus
  • Power supply
  • Gel electrophoresis system with transfer insert (Bio‐Rad Mini‐PROTEAN with Mini Trans‐Blot)
  • Gel imaging system capable of chemiluminescence detection

Basic Protocol 2: Photochemical Control of Nuclear Localization

  Materials
  • HeLa cells (ATCC)
  • Complete DMEM with antibiotics (Life Technologies; see recipe)
  • DMEM (see recipe)
  • OptiMEM (Life Technologies)
  • pPCKRS/PylT (available from Alexander Deiters, University of Pittsburgh or Jason W. Chin, Medical Research Council Laboratory of Molecular Biology; Gautier et al., )
  • pEGFP‐OptoNLS‐mCherry (available from Hanna Engelke, University of Munich; Engelke et al., )
  • Photocaged lysine (PCK) stock solution (100 mM) in water (see recipe)
  • Linear polyethylenimine (L‐PEI) transfection solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Phenol red‐free DMEM (Life Technologies)
  • 8‐well chamber slide (LabTek)
  • Biological safety cabinet
  • Fluorescence microscope with 40× objective, filter cube or laser for mCherry imaging, live‐cell incubation chamber, and 365‐nm LED (Lumen Dynamics) light source with DMD stimulation module (Nikon)
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Figures

Videos

Literature Cited

Literature Cited
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