Unit

UNIT 21.17 Applications for Chemical Probes of Proteolytic Activity

  1. Matthew Bogyo1,
  2. Amos Baruch2,
  3. Douglas A. Jeffery2,
  4. Doron Greenbaum3,
  5. Anna Borodovsky4,
  6. Huib Ovaa4,
  7. Benedikt Kessler4

Published Online: 1 SEP 2004

DOI: 10.1002/0471140864.ps2117s36

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Bogyo, M., Baruch, A., Jeffery, D. A., Greenbaum, D., Borodovsky, A., Ovaa, H. and Kessler, B. 2004. Applications for Chemical Probes of Proteolytic Activity. Current Protocols in Protein Science. 36:21.17:21.17.1–21.17.35.

Author Information

  1. 1

    Stanford University, Stanford, California

  2. 2

    Celera Genomics, South San Francisco, California

  3. 3

    University of California, San Francisco, California

  4. 4

    Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 SEP 2004
  2. Published Print: MAY 2004

Abstract

Recent genome sequencing projects have identified new peptidases in multiple organisms, many with unknown functions, suggesting the need for new tools to study these enzymes. This unit outlines selection and use of small-molecule and protein-based probes to covalently modify peptidases in complex cellular environments. These activity-based probes (ABPs) have been designed based on well characterized peptidase inhibitor scaffolds, but make use of new techniques to greatly enhance their utility for studying families of related peptidases. In particular, ABPs can be used to track activity of peptidases in crude cell extracts, intact cells, and in vivo, allowing rapid purification and identification of labeled targets. They can be used with libraries of small molecules to rapidly assess potency and selectivity of compounds in complex, physiologically relevant samples. Probe selection, probe tagging using reporters, labeling of recombinant targets, crude protein extracts, and peptidase targets in cell culture systems, affinity purification of targets, and inhibitor screening using affinity probes are outlined.

Keywords:

  • affinity labels;
  • activity based probes;
  • cysteine peptidases;
  • serine peptidases;
  • DUBs;
  • affinity purification;
  • inhibitor competition;
  • inhibitor selectivity