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

  • Unit Introduction
  • Strategic Planning
  • Basic Protocol: Fluorescent Assay of Site-Specific Protease
  • Support Protocol: Substrate-GFP Fusion Protein Purification Using Metal-Chelate Chromatography
  • Alternate Protocol: Fluorescent Resonance Energy Transfer Assay of Site-Specific Proteases
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
     
 
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Materials

Basic Protocol: Fluorescent Assay of Site-Specific Protease

 Materials
  • Purified stock substrate-GFP fusion protein (see Support Protocol)
  • 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
  • 5× 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: 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)
  • 5× binding buffer (see recipe)
  • 1:1 (w/v) aqueous slurry of Talon resin
  • Elution 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

  •  FigureFigure 21.9.1 Flow diagram of fluorescent sequence-specific protease assay.
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  •  FigureFigure 21.9.2 Rate of GFP release from the decapeptide containing recombinant polyprotein, SSU-10-GFP, and the recombinant polyprotein, SSU-GFP, lacking the decapeptide. Substrates were incubated with chloroplast extract and at the indicated times, uncleaved GFP containing substrate was bound to metal-chelate resin. The resin was recovered by centrifugation and unbound GFP remaining in the supernatant was quantitated by spectrofluorometry.
    View Image
  •  FigureFigure 21.9.3 Western blot of GFP released from the decapeptide containing recombinant polyprotein, SSU-10-GFP, and the recombinant polyprotein, SSU-GFP, lacking the decapeptide. Substrates were incubated with chloroplast extract and at the indicated times, uncleaved GFP containing substrate was bound to metal-chelate resin. The resin was recovered by centrifugation and unbound proteins remaining in the supernatant were separated by SDS-polyacrylamide gel electrophoresis. Proteins were blotted to nitrocellulose and GFP containing proteins were detected using anti-GFP as the primary antibody and horseradish peroxidase as the secondary antibody.
    View Image

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.

This work was supported by National Science Foundation grants MCB 9630817 and MCB 00800345 (S.D.S.) and the Center for Biotechnology, University of Nebraska-Lincoln.

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