Use of GFP as a Reporter for the Analysis of Sequence‐Specific Proteases

Gautam Sarath1, Steven D. Schwartzbach2

1 University of Nebraska, Lincoln, Nebraska, 2 University of Memphis, Memphis, Tennessee
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.9
DOI:  10.1002/0471140864.ps2109s26
Online Posting Date:  February, 2002
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Abstract

This unit describes a rapid fluorescent assay for sequence‐specific proteases. A recombinant His‐tagged substrate‐GFP fusion protein containing the sequence‐specific protease‐recognition sequence is used as substrate. Batch metal‐chelate chromatography separates uncleaved substrate‐GFP fusion protein from GFP released by proteolysis and proteolytic activity is determined by measuring the fluorescence of GFP remaining in solution.

     
 
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Table of Contents

  • Basic Protocol 1: Fluorescent Assay of Site‐Specific Protease
  • Support Protocol 1: Substrate‐GFP Fusion Protein Purification Using Metal‐Chelate Chromatography
  • Alternate Protocol 1: Fluorescent Resonance Energy Transfer Assay of Site‐Specific Proteases
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Fluorescent Assay of Site‐Specific Protease

  Materials
  • Purified stock substrate‐GFP fusion protein (see protocol 2)
  • 5× or 10× stock buffers for protease assay
  • Stock solutions of additives for optimal enzyme activity
  • Extract containing sequence‐specific protease
  • Milli‐Q water or equivalent
  • recipe5× binding buffer (see recipe), ice cold
  • Washed metal‐chelate affinity resin, e.g., 1:1 (w/v) aqueous slurry of Talon beads (Clontech)
  • Stock solutions of sequence‐specific protease inhibitor (optional)
  • 1.7‐ml microcentrifuge tubes
  • Rocker‐shaker, 4°C
  • Spectrofluorometer/fluorescence plate reader

Support Protocol 1: Substrate‐GFP Fusion Protein Purification Using Metal‐Chelate Chromatography

  Materials
  • E. coli transformed with expression vector encoding His‐tagged substrate‐GFP fusion protein
  • LB medium (unit 5.2)
  • recipe5× binding buffer (see recipe)
  • 1:1 (w/v) aqueous slurry of Talon resin
  • recipeElution buffer (see recipe)
  • Enzyme assay buffer
  • 26°C rotary shaker for growing bacterial cultures
  • 15‐ml culture tubes
  • 2‐liter flasks
  • Spectrofluorometer
  • 250‐ml centrifuge bottles
  • Refrigerated centrifuge
  • 50‐ml conical centrifuge tubes
  • Sonicator with microtip
  • 26‐ml polycarbonate bottles for ultracentrifuge
  • Ultracentrifuge with a 70 Ti rotor (Beckman) or equivalent
  • 1‐ml gravity flow chromatography columns
  • Additional reagents and equipment for dialysis ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
   Chan, R.L., Keller, M., Canaday, J., Weil, J.H., and Imbault, P. 1990. Eight small subunits of Euglena ribulose 1‐5 bisphosphate carboxylase/oxygenase are translated from a large mRNA as a polyprotein. EMBO J. 9:333‐338.
   Enomoto, T., Sulli, C., and Schwartzbach, S.D. 1997. A soluble chloroplast protease processes the Euglena polyprotein precursor to the light harvesting chlorophyll a/b binding protein of photosystem II. Plant Cell Physiol. 38:743‐746.
   Houlne, G. and Schantz, R. 1988. Characterization of cDNA sequences for LHCI apoproteins in Euglena gracilis: The mRNA encodes a large precursor containing several consecutive divergent polypeptides. Mol. Gen. Genet. 213:479‐486.
   Mitra, R.D., Silva, C.M., and Youvan, D.C. 1996. Fluorescence resonance energy transfer between blue‐emitting and re‐shifted excitation derivatives of the green fluorescent protein. Gene 173:13‐17.
   Muchhal, U. and Schwartzbach, S.D. 1992. Characterization of a Euglena gene encoding a polyprotein precursor to the light harvesting chlorophyll a/b binding protein of photosystem II. Plant Mol. Biol. 18:287‐299.
   Pehrson, J.C., Weatherman, A., Markwell, J., Sarath, G., and Schwartzbach, S.D. 1999. The use of GFP for the facile analysis of sequence‐specific proteases. BioTechniques 27:28‐32.
   Schiff, J.A., Schwartzbach, S.D., Osafune, T., and Hase, E. 1991. Photocontrol and processing of LHCPII apoprotein in Euglena: Possible role of golgi and other cytoplasmic sites. J. Photochem. Photobiol. Biol. 11:219‐236.
   Stryer, L. 1978. Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47:819‐846.
   Tsien, R.Y. 1998. The green fluorescent protein. Annu. Rev. Biochem. 67:509‐544.
Key References
   Enomoto et al., 1997. See above.
  Demonstrates the difficulties inherent in using radiolabeled substrates produced by in vitro translation and SDS gel electrophoresis to assay site‐specific protease activity.
   Tsien 1998. See above.
  A comprehensive review of the biochemical properties of GFP and its uses in biological research.
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