Reconstitution of Human CCR4-NOT Complex from Purified Proteins and an Assay of Its Deadenylation Activity

Methods Mol Biol. 2024:2723:1-17. doi: 10.1007/978-1-0716-3481-3_1.

Abstract

We describe protocols to produce and reconstitute an active human CCR4-NOT complex. Individual recombinant subunits are expressed in E. coli or baculovirus-infected insect cells, purified using column chromatography, and reconstituted into a stable complex containing all eight core subunits. In addition, we describe the biochemical assay of deadenylation using the reconstituted complex.

Keywords: CCR4-NOT; Deadenylation; Reconstitution; mRNA decay; poly(A) tail.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Escherichia coli* / genetics
  • Escherichia coli* / metabolism
  • Exoribonucleases* / metabolism
  • Humans
  • RNA Stability
  • Receptors, CCR4
  • Ribonucleases / metabolism
  • Transcription Factors / metabolism

Substances

  • Exoribonucleases
  • Transcription Factors
  • Ribonucleases
  • CCR4 protein, human
  • Receptors, CCR4