Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin

Protein Expr Purif. 2016 Mar;119:117-23. doi: 10.1016/j.pep.2015.11.020. Epub 2015 Nov 28.

Abstract

Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

Keywords: Affinity chromatography; Molecular chaperones; Protein–protein interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / isolation & purification*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / isolation & purification*
  • Cell Extracts / isolation & purification
  • Chaperonin 60 / chemistry
  • Chaperonin 60 / isolation & purification*
  • Chaperonin 60 / metabolism
  • Chromatography, Affinity
  • Escherichia coli
  • Male
  • Mitochondria, Liver / metabolism
  • Oxidative Stress
  • Protein Binding
  • Protein Denaturation
  • Rats, Wistar

Substances

  • Archaeal Proteins
  • Bacterial Proteins
  • Cell Extracts
  • Chaperonin 60