LOV Domains as In Vivo Fluorescent Reporters of Protein Expression

John M. Christie1

1 Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 13.1
DOI:  10.1002/9780470089941.et1301s06
Online Posting Date:  September, 2012
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Abstract

Chromophore‐binding domains from plant and bacterial photoreceptors have received increasing interest as new sources of genetically encoded fluorescent proteins (FPs). In particular, FPs based on the flavin‐binding LOV (Light, Oxygen, or Voltage sensing) domain offer advantages over green fluorescent protein (GFP) owing to their smaller size and utility under anaerobic conditions. Recombinant expression of LOV domains in Escherichia coli (E. coli) is fast, easy to detect, and inexpensive given the innate ability of LOV domains to acquire their ubiquitous organic cofactor from the cellular environment. This manuscript describes the strategies and variables to consider when expressing and purifying LOV‐domain protein fusions from liquid cultures of E. coli. Strategies for expressing and visualizing LOV‐domain fusion proteins in E. coli grown on agar medium are also described. Curr. Protoc. Essential Lab. Tech. 6:13.1.1‐13.1.11. © 2012 by John Wiley & Sons, Inc.

Keywords: LOV domain; FMN; flavin mononucleotide; protein expression; E. coli; fusion protein; soluble protein; affinity purification; UV‐A/blue light; fluorescence spectroscopy

     
 
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Table of Contents

  • Introduction
  • Protocols
  • Basic Protocol 1: Plasmid Preparation and Cloning a Gene of Interest
  • Basic Protocol 2: Visualization of iLOV Expression in E. coli Colonies
  • Basic Protocol 3: Expression and Quantification of iLOV Fluorescence in Liquid Cultures
  • Basic Protocol 4: Purification and Visualization of iLOV‐Tagged Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Plasmid Preparation and Cloning a Gene of Interest

  Materials
  • Plasmid vector pET28a‐iLOV (Fig. ); available upon request from the author (john.christie@glasgow.ac.uk)
  • Chemically competent E. coli strain DH5α (e.g., Invitrogen)
  • LB agar plates (see recipe) containing 50 µg/ml kanamycin
  • NEBuffer 4 (New England Biolabs)
  • 100× bovine serum albumin (BSA; New England Biolabs)
  • Restriction enzymes: NotI‐HF and XhoI (New England Biolabs)
  • 10 U/µl calf intestinal alkaline phosphatase (New England Biolabs)
  • QIAquick DNA Extraction and PCR Purification Kits (Qiagen)
  • Gene sequence of interest (plasmid carrying cDNA encoding the protein to be expressed)
  • PCR primers for cloning into pET28a‐iLOV containing the appropriate restriction sites (see Fig. )
  • High‐fidelity thermostable DNA polymerase: e.g., 2.5 U/µl PfuUltra and 10× PfuUltra buffer (Agilent)
  • 5 mM dNTP mix (unit 3.3)
  • 0.8% to 1% agarose gel (unit 7.2)
  • 400 U/µl T4 DNA ligase and 10× T4 ligase buffer (New England Biolabs)
  • 65°C water bath or heat block
  • Thermal cycler (see unit 10.2)
  • Additional reagents and equipment for transforming E. coli (Seidman et al., ), culture of E. coli (unit 4.2), DNA minipreps (Engebrecht et al., ), determination of DNA concentration (unit 2.2), the polymerase chain reaction (PCR; unit 10.2), agarose gel electrophoresis (unit 7.2), and DNA sequencing (Ausubel et al., , Chapter 7).

Basic Protocol 2: Visualization of iLOV Expression in E. coli Colonies

  Materials
  • pET28a‐iLOV containing gene of interest ( protocol 1)
  • Positive control: pET28a‐iLOV (Fig. ); available upon request from the author (john.christie@glasgow.ac.uk)
  • Negative control: pET28a (Novagen)
  • Chemically competent E. coli strain BL21(DE3) (Invitrogen or Novagen)
  • LB agar plates (see recipe) containing 50 µg/ml kanamycin and 100 µg/ml isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
  • Handheld long‐wave UV lamp (365 nm)
  • Additional reagents and equipment for transforming E. coli (Seidman et al., ) and culture of E. coli (unit 4.2)

Basic Protocol 3: Expression and Quantification of iLOV Fluorescence in Liquid Cultures

  Materials
  • pET28a‐iLOV containing gene of interest ( protocol 1)
  • Positive control: pET28a‐iLOV (Fig. ); available upon request from the author (john.christie@glasgow.ac.uk)
  • Negative control: pET28a (Novagen)
  • Chemically competent E. coli strain BL21(DE3) (Invitrogen or Novagen)
  • LB liquid medium (see recipe) containing 50 µg/ml kanamycin
  • 0.4 M isopropyl‐β‐D‐thiogalactopyranoside (IPTG)
  • Handheld long‐wave UV lamp (365 nm; e.g., Spectroline from UVP)
  • Fluorescence spectrometer
  • Absorbance spectrometer measuring in visible range
  • Additional reagents and equipment for transforming E. coli (Seidman et al., ) and culture of E. coli (unit 4.2)

Basic Protocol 4: Purification and Visualization of iLOV‐Tagged Proteins

  Materials
  • E. coli transformed with pET28a‐iLOV containing the gene of interest ( protocol 1, step 3)
  • LB medium (see recipe) containing 50 µg/ml kanamycin
  • 0.4 M isopropyl‐β‐D‐thiogalactopyranoside (IPTG)
  • Lysis buffer (see recipe)
  • Ni‐NTA agarose (Qiagen)
  • Elution buffer (see recipe)
  • Sonicator (e.g., MSE Soniprep 150 fitted with an exponential microprobe)
  • Refrigerated centrifuge
  • Disposable chromatography column (0.8 × 4–cm Poly‐Prep chromatography column; Bio‐Rad; also see unit 6.2)
  • Handheld long‐wave UV lamp (365 nm)
  • Additional reagents and equipment for culture of E. coli (unit 4.2), column chromatography (unit 6.2), and SDS‐PAGE (unit 7.3)
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Figures

Videos

Literature Cited

Literature Cited
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2012. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
   Chapman, S., Faulkner, C., Kaiserli, E., Garcia‐Mata, C., Savenkov, E.I., Roberts, A.G., Oparka, K.J., and Christie, J.M. 2008. The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection. Proc. Natl. Acad. Sci. U.S.A. 105:20038‐20043.
   Christie, J.M. 2007. Phototropin blue‐light receptors. Annu. Rev. Plant. Biol. 58:21‐45.
   Christie, J.M., Corchnoy, S.B., Swartz, T.E., Hokenson, M., Han, I.S., Briggs, W.R., and Bogomolni, R.A. 2007. Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1. Biochemistry 46:9310‐9319.
   Drepper, T., Eggert, T., Circolone, F., Heck, A., Krauss, U., Guterl, J.K., Wendorff, M., Losi, A., Gartner, W., and Jaeger, K.E. 2007. Reporter proteins for in vivo fluorescence without oxygen. Nat. Biotechnol. 25:443‐445.
   Engebrecht, J., Brent, R. and Kaderbhai, M. A. 1991. Minipreps of plasmid DNA. Curr. Protoc. Mol. Biol. 15:1.6.1–1.6.10.
   Seidman, C. E., Struhl, K., Sheen, J. and Jessen, T. 1997. Introduction of plasmid DNA into cells. Curr. Protoc. Mol. Biol. 37:1.8.1–1.8.10.
   Shaner, N.C., Patterson, G.H., and Davidson, M.W. 2007. Advances in fluorescent protein technology. J. Cell. Sci. 120:4247‐4260.
   Shu, X., Lev‐Ram, V., Deerinck, T.J., Qi, Y., Ramko, E.B., Davidson, M.W., Jin, Y., Ellisman, M.H., and Tsien, R.Y. 2011. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9:e1001041.
   Tsien, R.Y. 1998. The green fluorescent protein. Annu. Rev. Biochem. 67:509‐544.
   Tsien, R.Y. 2009. Constructing and exploiting the fluorescent protein paintbox (Nobel Lecture). Angew Chem. Int. Ed. Engl. 48:5612‐5626.
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