Engineering caspase 7 as an affinity reagent to capture proteolytic products

FEBS J. 2021 Feb;288(4):1259-1270. doi: 10.1111/febs.15467. Epub 2020 Jul 11.


Many proteases recognize their substrates with high specificities, with this in mind, it should theoretically be possible to utilize the substrate binding cleft of a protease as a scaffold to engineer an affinity reagent. In this study, we sought to develop reagents that would differentiate between substrates and products of proteolysis, based on a caspase 7 scaffold. Firstly, we engineered a form of caspase 7 that can undergo conversion to a substrate binding conformation without catalysis. Seeking to generate a product-only trap, we further engineered this construct by incorporating mutations that compensate for the generation of a negative charge in the neo C terminus of a newly generated product. This was accomplished with only three substitutions within the substrate binding cleft. Moreover, the affinity of the product trap for peptides was comparable to the affinity of caspase 7 to parental substrates. Finally, generation of a hybrid fluorescent protein with the product trap provided a reagent that specifically recognized apoptotic cells and highlights the versatility of such an approach in developing affinity and imaging agents for a variety of cysteine and serine proteases.

Keywords: apoptosis; mutagenesis; protease trap; protein engineering; proteolysis imaging.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects
  • Binding Sites / genetics
  • Caspase 7 / chemistry
  • Caspase 7 / genetics*
  • Caspase 7 / metabolism
  • Cell Line, Tumor
  • Endopeptidases / metabolism
  • Humans
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism*
  • Mutation*
  • Protein Binding
  • Protein Domains
  • Protein Engineering / methods*
  • Proteolysis
  • Substrate Specificity
  • TNF-Related Apoptosis-Inducing Ligand / pharmacology


  • Mutant Proteins
  • TNF-Related Apoptosis-Inducing Ligand
  • Endopeptidases
  • TEV protease
  • Caspase 7