Disassembly/reassembly strategy for the production of highly pure GroEL, a tetradecameric supramolecular machine, suitable for quantitative NMR, EPR and mutational studies

Protein Expr Purif. 2018 Feb;142:8-15. doi: 10.1016/j.pep.2017.09.010. Epub 2017 Sep 22.

Abstract

GroEL, a prototypical member of the chaperonin class of chaperones, is a large supramocular machine that assists protein folding and plays an important role in proteostasis. GroEL comprises two heptameric rings, each of which encloses a large cavity that provides a folding chamber for protein substrates. Many questions remain regarding the mechanistic details of GroEL facilitated protein folding. Thus, data at atomic resolution of the type provided by NMR and EPR are invaluable. Such studies often require complete deuteration of GroEL, uniform or residue specific 13C and 15N isotope labeling, and the introduction of selective cysteine mutations for site-specific spin labeling. In addition, high purity GroEL is essential for detailed studies of substrate-GroEL interactions as quantitative interpretation is impossible if the cavities are already occupied and blocked by other protein substrates present in the bacterial expression system. Here we present a new purification protocol designed to provide highly pure GroEL devoid of non-specific protein substrate contamination.

Keywords: Disassembly/reassembly; GroEL; Purification for biophysical studies; Supramolecular machine.

MeSH terms

  • Ammonium Sulfate / chemistry
  • Chaperonin 60 / chemistry
  • Chaperonin 60 / genetics
  • Chaperonin 60 / isolation & purification*
  • Chaperonin 60 / metabolism
  • Chromatography, Gel / methods*
  • Chromatography, Ion Exchange / methods*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / isolation & purification*
  • Escherichia coli Proteins / metabolism
  • Gene Expression
  • Nitrogen Isotopes / chemistry
  • Nuclear Magnetic Resonance, Biomolecular
  • Point Mutation*
  • Protein Multimerization
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Streptomycin / chemistry
  • Urea / chemistry

Substances

  • Chaperonin 60
  • Escherichia coli Proteins
  • Nitrogen Isotopes
  • Recombinant Proteins
  • Urea
  • Ammonium Sulfate
  • Streptomycin