Microsphere‐Based Flow Cytometry Protease Assays for Use in Protease Activity Detection and High‐Throughput Screening

Matthew J. Saunders1, Bruce S. Edwards2, Jingshu Zhu3, Larry A. Sklar2, Steven W. Graves1

1 National Flow Cytometry Resource, Los Alamos National Laboratory, Los Alamos, New Mexico, 2 University of New Mexico Center for Molecular Discovery, Albuquerque, New Mexico, 3 Center for Biomedical Engineering, Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.12
DOI:  10.1002/0471142956.cy1312s54
Online Posting Date:  October, 2010
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This protocol describes microsphere‐based protease assays for use in flow cytometry and high‐throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein‐specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high‐throughput analysis and inhibitor screening. Curr. Protoc. Cytom. 54:13.12.1‐13.12.17. © 2010 by John Wiley & Sons, Inc.

Keywords: protease assays; microspheres; high‐throughput; protease; multiplex microspheres; green fluorescent protein

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Microsphere‐Based Protease Assays by Flow Cytometry
  • Support Protocol 1: Growth and Purification of Biotinylated Proteins from Bacteria
  • Support Protocol 2: Specific Microsphere Binding Measurements for Optimal Display of Protease Substrates
  • Support Protocol 3: Adaptation of Microsphere‐Based Protease Assays to High‐Throughput Screening by Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Microsphere‐Based Protease Assays by Flow Cytometry

  • Protease buffer (see recipe)
  • Microspheres functionalized with streptavidin or avidin at a stock concentration between 106 and 109/ml (Spherotech)
  • Purified and dialyzed biotinylated fluorescent protease substrate (see protocol 2)
  • Purified or purchased protease of interest
  • 1.5‐ml microcentrifuge tubes
  • A mixing device such as a rotator or Nutator
  • Vortex
  • A microcentrifuge capable of spinning microcentrifuge tubes between 13,000 and 16,000 × g
  • A flow cytometer capable of measuring EGFP fluorescence (e.g., with excitation at 488 nm and measuring 505 to 550 nm fluorescence)
NOTE: If multiplex microsphere sets are used, the cytometer must be able to detect the fluorescence channel of microsphere fluorescence, as well as substrate fluorescence.

Support Protocol 1: Growth and Purification of Biotinylated Proteins from Bacteria

  • Plasmid containing a biotinylation sequence on one end (Pinpoint from Promega, or AviTag vectors from Avidity LLC., Gateway plasmids from Life Technologies) and a fluorescent protein on the other end (e.g., GFP, YFP, etc.), cloned in‐frame with protease substrate sequence in between
  • Calcium‐competent or electrocompetent expression bacteria, which will biotinylate protein sequences in vivo
  • LB agar plates with 50 µg/ml carbenicillin/ampicillin and 34 µg/ml chloramphenicol
  • Terrific broth (Fisher Scientific)
  • Appropriate resistance antibiotic for plasmid selection (carbenicillin or ampicillin for Pinpoint plasmids and chloramphenicol for the strains used here)
  • Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG; Sigma‐Aldrich)
  • Phosphate‐buffered saline, pH 7.2 (PBS; Sigma, cat. no. P3813‐10PAK)
  • Softlink streptavidin resin (Promega)
  • Biotin (Sigma‐Aldrich)
  • Protease buffer (see recipe)
  • Column chromatography system or peristaltic pump with glass column
  • Ultra centrifugal filter units (MWCO of appropriate size based on the protein used; Millipore)
  • 37°C incubator
  • Spectrophotometer capable of measuring absorbance values at a wavelength of 600 nm
  • Centrifuge capable of forces up to 25,000 × g
  • Centrifuge tubes and rotors to sediment 200 to 500 ml culture and 30 to 50 ml bacterial lysate
  • Dialysis membrane with appropriate MWCO values Dialysis clamps
  • Additional reagents and equipment for transforming E. coli (Seidman et al., )
NOTE: Biotinylated protein purifications have been done using BL21 (DE3) pLys S bacteria available from Promega. Additional specific biotinylation can be achieved by using the AVB 101 strain available from Avidity LLC.

Support Protocol 2: Specific Microsphere Binding Measurements for Optimal Display of Protease Substrates

  • Phosphate‐buffered saline, pH 7.2 (PBS; Sigma, cat. no. P3813‐10PAK)
  • Streptavidin‐ or avidin‐coated microspheres, either prepared in lab or commercially purchased (Spherotech)
  • Purified protease substrate biotinylated on one terminus with a fluorophore for detection on the other terminus ( protocol 2)
  • Biotin (Sigma‐Aldrich)
  • 1.5‐ml microcentrifuge tubes
  • Mixing device such as a rotator or neutator
  • Flow cytometer capable of excitation and detection at appropriate wavelength for substrate fluorophore (488 nm excitation for GFP, detection in 505 to 550 nm range)

Support Protocol 3: Adaptation of Microsphere‐Based Protease Assays to High‐Throughput Screening by Flow Cytometry

  • Purified fluorescent protease substrate with biotin at one end and a fluorophore at the other end with the protease substrate or cleavage site in between ( protocol 2)
  • Streptavidin‐ or avidin‐coated microspheres at a stock concentration of 106 to 109 per ml
  • Protease buffer (see recipe)
  • Test compounds (chemical libraries, etc.)
  • Purified or purchased protease of interest
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge capable of spinning 1.5‐ml tubes between 13,000 and 16,000 × g
  • A flow cytometer with appropriate excitation lasers and emissions filters for excitation and detection of fluorophore incorporated in protease substrate
  • 96‐ or 384‐well microplates
  • Multichannel pipets
  • Rotating device
  • Plate acquisition device for flow cytometry (e.g., HyperCyt, BD LSR II HTS, or other)
  • Data analysis software (e.g., FlowJo, FCS Express, IDL Hyperview, or any other flow cytometry data analysis software)
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Literature Cited

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