Efficient recovery of a functional extracellular domain of bovine IgG2 Fc receptor (boFcgamma2R) from inclusion bodies by a rapid dilution refolding system

J Immunol Methods. 2008 May 20;334(1-2):21-8. doi: 10.1016/j.jim.2008.01.020. Epub 2008 Mar 3.

Abstract

The extracellular domain of the boFcgamma2R gene was constructed and cloned into the Escherichia coli expression vector pET-28a. The recombinant protein was expressed at high level in E. coli BL21(DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine hydrochloride and refolded by rapid dilution. After renaturation, the purity of the recovered recombinant protein was up to 95%. ELISA assay showed that the renatured recombinant protein could inhibit bovine IgG2 binding to expressed boFcgamma2R on the COS-7 cell surface with an IC50 value of 0.68 microM. The overall yield of the active rsbo2R was up to 20 mg/l of culture. Crystals of the rsbo2R were grown at 293 K by the hanging-drop vapour diffusion method showed weak diffraction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Cattle
  • Chlorocebus aethiops
  • Cloning, Molecular
  • Crystallization / methods
  • Enzyme-Linked Immunosorbent Assay
  • Immunoglobulin G / immunology*
  • Inclusion Bodies / immunology*
  • Protein Folding
  • Protein Renaturation
  • Receptors, IgG / chemistry
  • Receptors, IgG / immunology
  • Receptors, IgG / isolation & purification*
  • Receptors, IgG / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / immunology
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • Immunoglobulin G
  • Receptors, IgG
  • Recombinant Proteins