A ubiquitin-based tagging system for controlled modulation of protein stability

Nat Biotechnol. 2000 Dec;18(12):1298-302. doi: 10.1038/82422.

Abstract

Many biotechnology applications depend on the expression of exogenous proteins in a predictable and controllable manner. A key determinant of the intracellular concentration of a given protein is its stability or "half-life." We have developed a versatile and reliable system for producing short half-life forms of proteins expressed in mammalian cells. The system consists of a series of destabilization domains composed of varying numbers of a mutant form of ubiquitin (UbG76V) that cannot be cleaved by ubiquitin hydrolases. We show that increasing the number of UbG76V moieties within the destabilization domain results in a graded decrease in protein half-life and steady-state levels when fused to heterologous reporter proteins as well as cellular proteins. Cells expressing a destabilized beta-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.

Publication types

  • Evaluation Study

MeSH terms

  • Biopolymers / genetics
  • Biopolymers / metabolism
  • Cysteine Endopeptidases / metabolism*
  • Enzyme Stability
  • Genes, Reporter
  • Half-Life
  • Humans
  • Jurkat Cells
  • Multienzyme Complexes / metabolism*
  • Plasmids / genetics
  • Polyubiquitin
  • Proteasome Endopeptidase Complex
  • Protein Conformation
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • Ubiquitins / chemistry*
  • Ubiquitins / genetics
  • Ubiquitins / metabolism*
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism

Substances

  • Biopolymers
  • Multienzyme Complexes
  • Recombinant Fusion Proteins
  • Ubiquitins
  • Polyubiquitin
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex
  • beta-Lactamases